Shirou Emiya is first drawn into the Holy Grail War after he witnesses the Servants Lancer and Archer fighting at his school. Lancer chases Shirou around the school trying to kill him, adhering to a rule of the Holy Grail War which states bystanders must be killed. Lancer corners Shirou in one of the hallways and mortally wounds him with his Gáe Bolg. Rin Tohsaka, Shirou's classmate and Archer's master, feels guilty about involving Shirou and saves his life using her strongest jewel. Before Lancer can kill him, Shirou summons Saber, who quickly drives Lancer away using her invisible sword. She then attacks Archer and Rin, who are outside the house, and badly wounds Archer. Saber starts to attack Rin, but Shirou, confused by the situation, stops Saber before she kills Rin. As they leave the church, a young German Master named Illyasviel von Einzbern assaults them with her Servant, Berserker. Berserker, whose true identity is the Greek hero Heracles, completely overwhelms Saber. He severely wounds her, but as he lets loose the finishing blow, Shirou jumps into its path, shielding Saber and taking the fatal blow. Ilya, confused by this action, calls off Berserker and leaves. Later, Shirou wakes up fully healed inside his home. He also sees Rin there and learns she aided him again, caring for him as he recovered. Shirou and Rin agree to form an alliance in order to protect themselves from the pair of Ilya and Berserker, an immediate threat to their lives. The reasoning behind this alliance is simple - each of their servants are operating at a sub-optimal capacity; as part of an incomplete summon, Saber cannot use her full power; likewise, Archer is still recovering from the wounds he received while clashing with Saber. During the course of this alliance, they discover a spell has been placed on the school, one that will drain the life force of anyone within its range.

Shirou soon discovers, much to his dismay, that the Master who placed the life-draining spell over the school was Shinji. This revelation is enough to force a confrontation between the two. By this time, Shirou has begun to excel at reinforcement magic and melee fighting, a fact which quickly becomes apparent as Shinji's spells prove useless against his foe. However, as Shirou prepares to deliver the finishing blow, Rider intervenes by pushing Shirou out a nearby window. While falling, Shirou uses a Command Spell to summon Saber, who rescues him. Saber then goes after Rider, who is attempting to use her Noble Phantasm to retreat. After a chase, the two clash atop a skyscraper. Rider attacks Saber with her mount, Pegasus, forcing Saber to reveal her own Noble Phantasm, Excalibur. With Excalibur revealed, Saber makes short work of Rider, defeating her with one blow. After this event, Shirou begins having visions each morning; the first is of Saber in her past role as Artoria, a lonely monarch who devoted herself solely to fighting for her country. The second vision is of himself, being saved by his adoptive father Kiritsugu Emiya, in a fire. Shirou's memories of his father makes him determined to become a hero, someone who can save people just as his father did; thus, he finds that he and Saber have more in common than he thought.

Soon afterward, the Emiya residence is attacked by Caster. Both Shirou and Saber go outside to fight her, but Caster is killed by someone wearing golden armor before a battle can begin. Saber identifies the killer as an Archer-class Servant. She remembers him as an individual from the previous Grail War, which was ended before its completion because the Holy Grail had been destroyed. Shirou learns that Saber was a combatant in the previous war as well; furthermore, she had been the Servant of his adoptive father and had, under his father's orders, destroyed the Grail. It is also revealed that she became a Servant shortly before her death and that her greatest desire remains to return to her life and relive it. These revelations bother Shirou, as he doesn't like the idea of her dying and redoing her life.

After this event, Kotomine decides to go into active action; he kidnaps Ilya, initiating a final confrontation. Shirou and his allies discover that Ilya was kidnapped because she is the vessel of the Grail - the embodiment of its power. Rin is then heavily wounded by Kotomine, rendering her unable to fight alongside Saber and Shirou. Shirou decides to return Avalon, Saber's final Noble Phantasm which had been inside of Shirou's body up to this point, to her. After this, Saber confronts Gilgamesh, while Kotomine and Shirou face off. Both Shirou and Saber use Avalon simultaneously, nullifying their enemies' attacks, and then proceed to defeat them. With no opposition remaining, the pair destroy the contents of the Grail, ensuring the evil within it will never be used again.

As a result, Saber vanishes to her original time. She returns as Artoria, and accepts her past without the wish to change it. At the end of this route, she weakly smiles and tells Sir Bedivere (who is attending her) "I think I will sleep much longer this time...".

Shortly after repelling Lancer, Saber prepares to attack Archer who approaches. However, Shirou uses his Command Spell to negate that action and ends up conversing with Rin. In the end, no alliance is made at the time. After introducing Shirou to the Holy Grail War rules from Kirei, Illyasviel von Einzbern attacks them with her Servant Berserker on their way home. Unlike in the Fate route, Archer briefly participates in the battle and his help proves to be vital in driving Berserker away. The next day, Shirou goes to school with Saber, introducing her to Taiga and Sakura. The next day, when all other students and teachers leave the school, Rin starts her assault on Shirou, relentlessly pursuing him with the intent to kill him unless he surrenders Saber's Command Spells. She is interrupted at the last moment by the sudden appearance of Rider, who attacks a student. Shirou and Rin promptly team up to drive Rider away. Shirou catches a quick glimpse of Shinji as they do.

Rin apologizes to Shirou for Archer's actions and tells him that she used a Command Spell to order Archer to never attack Shirou. During strategy planning, Rider activates Blood Fort Andromeda. However, when Shirou and Rin approach her, they find her already dead. The terrified Shinji runs off to the Kotomine Church, where Kirei grants him the use of Gilgamesh. In the meantime, Archer and Saber debate over how Rider was killed; while Saber claims she died in honor protecting her Master, Archer claims she was a weakling, once again creating a rift between him and the rest. Rider's death makes Shirou suspect that there is another Master in the school, one other than Shinji. He suspects his friend Issei Ryuudou at first, but it turns out that the Master is their teacher Soichiro Kuzuki, who controls Caster. When Shirou and Rin ambush them, Kuzuki surprises them with his innate fighting abilities, enhanced by Caster's enchantment spells, which are able to cripple Saber. In order to make it easier to plan and strategize, Rin comes to stay at Shiro's house.

The next day, Rin takes Shirou on a date, bringing Saber along. On returning home, they find Caster holding Taiga Fujimura hostage. When Shirou tries to follow her demands, Caster releases Taiga and strikes Saber with the Rule Breaker, transferring Shirou's Command Spells to her. At Saber's urging, Shirou and Rin leave. With his Command Spells gone, Rin declares that she will fight Kuzuki and Caster with only Archer. However, Shirou insists on helping and follows them despite Rin's objections. Meanwhile, Caster tortures Saber in an effort to break her and make her submit completely. However, Saber's magic resistance proves to be an obstacle for Caster's Command Spell. Shirou follows Rin and witnesses her confronting Kuzuki and Caster in the Kotomine Church, their new base of operation. To her shock, Archer suddenly betrays Rin and attacks her. Shirou saves her and confessed his feelings for her, and although she is reluctant and embarrassed, she eventually thanks him and accepts his help again. With Archer gone, both decide to ask Ilya and Berserker for their help. Unfortunately, they arrive too late and find Shinji and Gilgamesh have gotten there first. Gilgamesh makes short work of Berserker, then rips Ilya's heart out. Both of them then leave the castle, leaving Shirou and Rin in despair until Lancer suddenly emerges and offers his help, which they accept.

The next day, Shirou, Saber and Lancer head to the Einzbern Castle, whereupon Archer reveals that he is an alternate future Emiya Shirou, the hero who is betrayed by the ideals he fought for, and now he seeks to create a time paradox by killing his past self. He considers this an act of mercy, not wanting Shirou to see his ideals betray and crush him like the way they crushed Archer. While Shirou fights, Lancer goes to rescue Rin. Kirei suddenly appears and orders Lancer to kill her. However, Lancer demands that Kirei use his Command Spell for such an order, and Kirei does, ordering Lancer to kill himself instead. Rin is about to submit to her fate as Kirei approaches to kill her, when suddenly Lancer stabs Kirei in the back using Gae Bolg. Shinji, now believing he is free to do whatever he likes to Rin, is chased off by a just-revived Lancer. After setting Rin free, Lancer activates his Ansuz rune, burning the room as he dies. Shirou faces off with Archer, finally convincing Archer that he is not taking the wrong path as he has no regrets about his decision, no matter what repercussions it may have in the future. With that, Archer is defeated. Gilgamesh then appears, attempting to kill Shirou because of his nature as a "Faker", but Archer pushes Shirou out of the path of Gilgamesh's attack, seemingly sacrificing himself He then retreats when the fire started by Lancer inflames the whole castle and destroys it. Gilgamesh catches up with Shinji and inserts Ilya's heart into his body, making him the new incarnation of the Holy Grail.

That night, Rin makes a contract with Shirou to give him mana so that he could battle Gilgamesh. Shirou, Rin and Saber reach Gilgamesh's new hideout, the Ryuudou Temple. Rin tries to pull Shinji out of the embodiment of the Grail while Shirou fights Gilgamesh and Saber is held back by Assassin. Shirou is able to take out Gilgamesh by using his own version of Unlimited Blade Works. However, Gilgamesh revives one last time to try and make him the new incarnation of the Holy Grail. Fortunately, Archer, who also appeared to help Rin at the same time, comes to Shirou's aid, finishing off Gilgamesh with an arrow to the head. Rin bids her Servant goodbye, as Archer tells her to take care of his younger self, hoping that her care of Shirou will change the future and grant his wish to save Shirou from his ideals. In the end, Rin finally sees Archer with a genuine smile, the same one as Shirou's.

Shirou has started to realize that his friend Sakura is growing into a beautiful woman, and he starts to feel attracted to her. He also meets Zouken Matou, Sakura's grandfather, for the first time. Zouken greets him warmly at the Matou house and thanks him for being a friend to his grandchildren. Rin also appears at the beginning, and is more hostile towards Shirou than in the Fate route. While patrolling at night with Saber, Shirou encounters Shinji and Rider. A fight breaks out between them, which Rider quickly loses. Though she has been defeated, Shinji abuses her as he orders her to get up despite her inability to, an act which even Saber condemns him for. Before she can act further, Zouken appears and tells Shinji to run away, calling him a failure. With nothing more to do at the scene, Shirou and Saber leave. Unknown to them, however, Shinji is somehow able to revive Rider.

Shirou later encounters Rin and Archer. Before a fight can break out, Zouken appears, along with Caster's reanimated corpse. Saber dispatches her, while Archer kills Zouken. They sense a strange shadow lurking nearby the whole time. During the day, Shirou encounters Ilya and a friendship begins to develop between them. However, unknown to them, Zouken has resurrected, after using his worms to devour a woman, and forming a contract with the True Assassin. The next night, Shirou and Saber decide to investigate the Ryuudou Temple further, but they are separated. Saber fights the True Assassin, but is also ambushed by the mysterious shadow and perishes. Enraged, Shiro tries to attack Zouken, only to be repelled by the True Assassin. Before the True Assassin can kill him, Rider suddenly appears and drives him away, displaying an enhanced level of strength and ability that surprises Shirou. When Shirou asks her about her motives, Rider reveals that she was ordered to keep Shirou alive before leaving the scene.

Later taken to the Kotomine Church, Shirou finds out that Kirei is performing surgery to prolong Sakura's life. When he leaves the church, he finds Archer, who presents him with the question of whether he wants to cling to his ideals or discard them. After consulting with Ilya, Shirou decides that he wants to protect Sakura. Thus, when Rin, who is adamant about killing Sakura before she causes any more harm, tries to visit Sakura, he stops her. She then reveals that Sakura is her sister. Before either can do anything further, they learn that Sakura has fled, and both pursue her. Shirou finds her first and embraces her, then takes her back to his house. Rin arrives soon after and declares their temporary alliance annulled, but Sakura surprisingly vows to fight back against her sister. Sakura and Rider are now living alongside Shirou at the Emiya residence. During this time, Sakura grows increasingly lustful and often asks Shirou to have sex with her. They later decide that they should warn Ilya about the impending danger from Zouken and from the mysterious shadow, which has begun to become more active at night. Shirou goes alone while Rider stays to guard Sakura, but Sakura later orders Rider to assist Shirou. In the forest, Shirou encounters Rin, but they are kept from engaging each other when they hear Berserker's roar. Zouken has attacked Ilya and Berserker with the True Assassin and Saber Alter, causing them to flee the castle. Berserker eventually falls, and the group escapes. However, the mysterious shadow attacks again, targeting Ilya. Shirou defends her, but their defense severs his left arm and mortally wounds Archer. Rider arrives too late to save him, but Archer tells her to transfer his left arm to Shirou.

In the meantime, Rin has been fighting Sakura and her army of shadows using the Gem Sword of Zelretch. During the course of the battle, Sakura continuously curses her sister for her ignorance towards her, telling her of the jealousy she feels. Rin initially brushes it off, but eventually lets herself be struck while she apologizes, hugs Sakura and expresses her true sisterly love. Realizing that her weakness allowed her to be consumed by her own darkness, Sakura goes berserk and loses control of her powers. Shirou arrives and attempts to save her. Despite Sakura's pleas to let her die, Shirou projects and stabs Sakura with Caster's Rule Breaker, severing Sakura's link to the unborn Servant within the Holy Grail, Avenger. A recovered Rider appears and escorts Rin and Sakura to safety as Shirou prepares to destroy the Great Holy Grail. Kirei suddenly appears, intending to stop Shirou from destroying that which he long desired to witness. They engage in hand-to-hand combat, in which Shirou eventually defeats Kirei. At this point the story diverges, based on whether the player headed to the Normal or True ending. On the Normal route, the story ends with Shirou destroying the Great Holy Grail with a projected Excalibur and dying, but on the True route path, Zouken, his consciousness still remaining in the last surviving worm, continues to struggle towards the grail. Before he can reach it, however, Ilya appears wearing the Dress of Heaven, which reminds Zouken of her ancestor, Justicia, who he loves, and he finally dies with satisfaction. The Great Holy Grail is destroyed. Sakura is the de facto winner of the 5th Holy Grail War in this route, seeing as all the other Masters are incapacitated or dead and all the Servants are defeated and dead, with exception of Rider. Even so, in his final moments Kotomine declares Shirou the winner of the war at the very end.