Convicted criminal Callum Lynch is rescued from his own execution by Abstergo Industries, the modern-day incarnation of theTemplar Order, and learns that they are searching for the Apple of Eden, which seeded the first sin, in order to subjugate the human race and end its perceived corruption. The company's head scientist, Dr. Sophia Rikkin, reveals that Callum is a descendant of Aguilar de Nerha, a member of a secret order of assassins that for centuries have opposed the Templar Order, and conscripts him to the Animus Project, in which Callum is connected to a machine that allows him to revive Aguilar's memories so Abstergo can learn the current whereabouts of the Apple of Eden.
In Spain, 1492, Aguilar and his partner Maria are deployed to rescue Prince Ahmed de Granada, who has been kidnapped by the leader of the Templars, Tomas de Torquemada, to coerce Prince Ahmed's father, Sultan Muhammad XII, to surrender the Apple of Eden. Aguilar and Maria intercept the chariot transporting Ahmed, but are then ambushed and captured by Torquemada's enforcer, Ojeda, just as Callum is pulled out of the Animus by Sophia.
In captivity, Callum befriends other descendants of assassins, led by Moussa, and begins experiencing hallucinations of both Aguilar and his own father, Joseph Lynch, who murdered Callum's mother while in assassin attire. Callum and Sophia build a rapport over their sessions, in which Sophia reveals herself as the daughter of Abstergo's CEO Alan Rikkin, and confides with Callum that her mother was likewise murdered by an assassin.
Back in the Animus, Aguilar and Maria are scheduled for execution, but Aguilar manages to free himself and Maria, leading to a rooftop chase in which they perform a Leap of Faith, and manage to escape. Callum is then extracted and placed back in captivity, where he learns from Joseph, who also present at the building, that his and Aguilar's memories are merging, allowing Callum to tap into Aguilar's combat abilities, and that Callum's mother was an assassin and chose to die by Joseph's hand rather than be subjected to the Animus by the Templars. Meanwhile, Alan is pressured by his superior Ellen Kaye to shut down the Animus Project due to its lack of results, leading Sophia to realize Alan cares more about the eradication of the assassins than using the Apple of Eden to rid the world of violence.
Callum is placed in the Animus once again, whereupon Aguilar and Maria ambush a meeting between Muhammad and Ojeda to exchange Ahmed for the Apple of Eden, killing the Templars and retrieving the Apple of Eden. Ojeda captures Maria to force Aguilar to surrender it, but Maria chooses death and Ojeda fatally stabs her before Aguilar kills him. Aguilar then escapes, performing another Leap of Faith, causing the Animus to malfunction. Aguilar then gives the Apple of Eden to Christopher Columbus, where it is found by Abstergo in his tomb. Rikkin and his followers then converge on a Templar sanctuary in London to perform a ritual to harness its powers, while Callum sees the hallucination of his mother and his ancestors, all in an assassin attire, fully embrace his assassin lineage.
Having assimilated all of Aguilar's memories and abilities, Callum manages to escape and rallies Moussa and the other descendants to stop the Templars. They attack the sanctuary and Callum comes across Sophia, who has grown disillusioned with the Templars and reluctantly allows them to act. Callum confronts Rikkin and silently kills him to retrieve the Apple of Eden, whereupon the assassins depart, vowing to once again protect it. Horrified by her father's death, Sophia vows revenge upon Callum.