Fate/Zero takes place 10 years prior to the events of Fate/stay night, centering around the events of the 4th Holy Grail War. Emiya Kiritsugu is the main protagonist of the story, hired by the Einzbern family to win the Grail War, Kiritsugu is married to Irisviel and is the Master of Saber. Meanwhile, Tokiomi Tohsaka, summons the Archer class Gilgamesh. Tokiomi is allied with the Church's overseer Risei Kotomine and his son, Kirei Kotomine, who becomes the Master of Assassin. Kariya Matou, after receiving grueling training from Zouken Matou, becomes the Master of Berserker. At the Clock Tower, Waver Velvet steals Lord Kayneth El-Melloi Archibald's artifact to summon Rider, Alexander the Great, to join the War, while Kayneth ends up summoning Lancer instead. Finally, a serial killer named Ryuunosuke Uryuu is chosen to become the War's final Master and summons Caster.

The story revolves around these seven Masters and their actions during the War, culminating in a final confrontation between Kiritsugu and Kirei. The decisions they make during this final confrontation is directly responsible for many events in the 5th Holy Grail War.