The game's first route features Saber as its heroine, and its completion is required to access the other two routes. The story begins with Rin summoning her Servant, Archer, whose identity is shrouded in mystery. At the school, Rin encounters Lancer and later Shirou, who unintentionally witnesses the fight between Lancer and Archer. Lancer decides to chase down and eliminate Shirou, but Shirou accidentally summons Saber, and thus becoming an unwilling participant of the Holy Grail War.

Rin and Shirou meet again later and agree to cooperate to defend themselves from the immediate threat, Berserker. However, they are off to a bad start: Saber can't use her full power because of Shirou's subpar magecraft abilities, and Archer is still recovering from the wound he received from Saber during their initial meeting.

While gathering information of the other Masters and Servants, they discover Matou Shinji, their classmate, as the master of Rider. Shinji, who can't use any magecraft himself, comes from a family of mages whose power died out down the generations. Shinji announces he doesn't want to fight Shirou and gives him information on another Servant residing in the Ryuudou Temple.

Shirou is not interested in picking a fight with that Servant, but Saber thinks differently, as she wants to defeat enemies that may be a threat to Shirou. Saber goes to the temple by herself, there she meets the Servant Assassin at the entrance. Before a fight breaks out, Shirou appears and stops them. Assassin decides to stop as well because he sensed a third enemy hiding behind the bushes, waiting for an opening.

A Master has been casting harmful spells around the school and endangering the life of innocent students. Shirou discovers Shinji to be the said Master, and for the first time Shirou is determined to fight another Master. By now Shirou is becoming more proficient using his enhancement magecraft and is skilled enough to hold his own in a fight. Shinji's low-level spells are useless to Shirou, when Shirou is about to win, Rider jumps in and knocks Shirou through the window. While falling down, Shirou uses his Command Seal to call Saber, who comes to his rescue. Rider uses her Noble Phantasm and escapes, but later they enter another fight in the New Town, on top of a building. Rider tries to push Saber down with her Pegasus, but Saber reveals her own Noble Phantasm, the Excalibur, and defeats Rider.

Shirou has been seeing two dreams every morning, one about a lonely king, Saber, who devoted everything to fight for her country. The king is King Arthur. The second dream is about him being saved by his foster father Kiritsugu in the fire. Shirou is determined to become someone who can save people just like Kiritsugu. He senses the same ideals in Saber.

When Shirou tries to befriend Ilya, he is instead kidnapped to her castle. Saber, Rin, and Archer go to rescue him. While escaping, Berserker and Ilya blocks their way. Archer fights by himself to let the others escape, leaving the famous last quote "By the way Rin, Can I ask you something?" "I don't mind buying time, but it wouldn't be a problem if I defeated him, would it?" Archer ends up defeating Berserker six times over before he died.

Saber, Shirou, and Rin escapes to a rundown shack, where by Rin's suggestion, Shirou and Saber have sex to replenish Saber's low mana supply. Having defeated Archer, Ilya and Berserker catch up to them. While a replenished Saber is able to fight Berserker equally, she can't hurt his iron body, whose head was even able to survive a surprise attack from Rin. Berserker's Noble Phantasm turns out to be an immortal body that can regenerate itself 12 times. When the battle seemed hopeless, Shirou awakens the true form of his magecraft, which was not Enhancement, but Tracing, an ability to create a mirror copy of an object he's seen with his own eyes. Shirou creates a copy of Saber's lost sword, Caliburn, that he saw in his dream, and with Saber's help, uses it to defeat Berserker. With Berserker gone Ilya is no longer able to participate in the War, so Shirou lets her stay in his house.

Shirou's house is attacked by Caster. When Shirou and Saber goes outside to confront her, a Servant in gold armor kills Caster. Saber calls this mysterious Servert "Archer." It turns out "Archer" was actually a Servant from the last Holy Grail War, which never ended properly because the Holy Grail was destroyed. Saber actually fought in the previous War as well, with Kiritsugu being her master. During the War's climax Kiritsugu used a Command Seal to force Saber to destroy the Holy Grail.

Shirou also finds out that Saber became a Servant right before she died, and her wish was to redo her life. Saber was said to be the greatest king ever, but ironically, people grew uncertain towards a king who never showed her human feelings. Saber's country eventually fell apart and she was standing over the corpses of people that she swore to protect. Saber wants to use the Holy Grail to redo her life where she never becomes a king, and makes a contract with the Holy Grail to become an Heroic Spirit.

Shirou doesn't like the idea of her dying and redoing her life, so he asks Kotomine, the Holy Grail War's overseer sent by the Church, if there is a way to keep her alive as a normal human. The answer is no, and the Holy Grail's wish-granting power is also put to question. Shirou takes Saber out to a date hoping to cheer her up, but it doesn't go too well and they are attacked by the gold Servant again, who is revealed to be Gilgamesh, the king of heroes. Shirou uses his body to shield Saber and barely survives thanks to Kiritsugu having implanted Avalon, sheath of Excalibur and Saber's last Noble Phantasm, within his body to save him back during the fire. This was also the reason Shirou was able to summon Saber.

Later Shirou goes to Kotomine's place again hoping to get more advice, but finds out Kotomine is the Master of not only Lancer, but Gilgamesh as well. Kotomine kept Gilgamesh from the last War by using orphans from the fire incident to supply Gilgamesh with mana, and obtained Lancer in this War by killing his original Master at the beginning. In the ensuing battle, Lancer, who just founded out he wasn't Kotomine's only Servant and is disgusted by his ways, sacrifices himself to help Shirou and Saber get away.

Before final battle, Kotomine kidnaps Ilya and reveals that she is actually the vessel of the Holy Grail. Rin is heavily wounded by Kotomine and is unable to fight alongside Shirou and Saber. To prepare, Shirou returns Avalon back to Saber. During the final battle, Saber fights Gilgamesh while Shirou confronts Kotomine. Both use Avalon at the same time, nullifying their opponents' attack, and defeat them. They realize the Holy Grail has been tainted, and decides to destroy it for good so it will be never used again. With the Grail War finally over, Saber decides to return to her time and end her life the way it was because she realized through Shirou that it is wrong for her to redo her life, especially for the many people she had sacrificed, Shirou respects her decision and sees her off. In Réalta Nua, an epilogue is added where Saber waits for Shirou inside Avalon after her death. Shirou believes he will see Saber again and endures many hardships in life. After he dies, Shirou and Saber are reunited in Avalon.