The game continues shortly after the events of Assassin's Creed, where, in 2012, Desmond Miles (Nolan North) is still a prisoner at Abstergo Industries after searching the memories of his ancestor, the Assassin, Altaïr, in the Animus. Abstergo, the modern-day equivalent of the Knights Templar, had been using Desmond to locate powerful out-of-place artifacts called "Pieces of Eden". Using the "Eagle Vision" ability he gained as a result of the "bleeding effect" (from overexposure to the Animus), Desmond is able to see messages and symbols written in blood on his bedroom wall, written by the former test subject, named "Subject 16". Lucy Stillman (Kristen Bell), a former researcher for Abstergo and a member of the modern-day Assassins, frees Desmond and brings him to a secret hideout for herself and two other Assassins, Shaun Hastings (Danny Wallace) and Rebecca Crane (Eliza Schneider). With their own version of the Animus, dubbed the 'Animus 2.0', they invite Desmond to help them discover the memories of his ancestor, Ezio Auditore da Firenze, for two reasons: to train Desmond in Assassin skills through the bleeding effect, and to find other Pieces of Eden.
Ezio, residing in Italy during the Renaissance in the late 15th century, finds his family in Florence the victims of a political plot, and is unable to prevent the hanging of his father and two brothers. His father's final instructions lead Ezio to a chest containing Assassin garments and tools. Ezio's uncle Mario allows Ezio and his mother and sister to shelter in his countryside villa, and trains Ezio in the art of combat. Mario further provides leads to those in on his family's betrayal, and Ezio follows the trail of clues, leading from Florence to San Gimignano, Forlì, Venice and eventually to Rome. Along the way, he encounters several allies that assist in his pursuit, including Leonardo da Vinci, who is able to help decode pages from Altaïr's Codex, creating new equipment. After assassinating many people involved in the plot, Ezio is able to identify Rodrigo Borgia as the leader of the conspiracy, aimed to bring down the Medici family. Borgia later arrives in Venice with the "Apple," the Piece of Eden that Altaïr originally had found. Borgia believes himself to be the "Prophet" that would lead to the discovery of "The Vault", supposedly containing more Pieces of Eden and other knowledge the Templars seek. Ezio, joined by his allies, stop Borgia, who flees without the Apple. His allies, including Niccolò Machiavelli, reveal they are all members of the Assassins, and bring Ezio into their ranks, telling him that they believe him to be the Prophet that will lead them to the Vault.
During one break from using the Animus, Desmond suffers a hallucination that returns him to the body of Altaïr in Acre, and makes love with Maria, a Templar he had earlier spared (a decoy for Robert de Sable in Assassin's Creed who Altaïr formed a relationship with in another adventure); when Altaïr departs, Desmond finds the memory staying with Maria, and leaves him confused when he recovers. He also learns of the case of Subject 16, one of several previous test subjects used by Abstergo, and the person responsible for writing the images he saw in his room at Abstergo. Subject 16 has managed to hack the Animus and place twenty glyphs that can only be seen and decoded by the Animus user, revealing a video of a man and a woman, claiming to be Adam and Eve, carrying the "Apple of Eden", running through a futuristic setting identified, by a flash of binary code, as Eden.
In the 'truth puzzles', certain historic paintings are used such as the first depiction of a fire arm.
When Desmond returns to Ezio's memory after he fought off Borgia, they find several corrupted memories, and are only able to return Desmond to Ezio's memories in the year 1499, where Borgia has since become Pope Alexander VI at Vatican City. Ezio's memories reveal that he and his allies have completed the Codex and discovered that "The Vault" lies in Rome, and realize that the Papal Staff is another Piece of Eden; Borgia's plan, upon deposition of the Medici family, would allow him to become Pope and gain access to the staff and the Vault.
Ezio and his allies travel to Rome; while his allies distract the rest of the city, Ezio discretely enters the Vatican and attempts to assassinate Borgia. Borgia manages to escape with both the Apple and the Staff, and attempts to use them to open the Vault, but Ezio catches up and defeats him but does not kill Borgia. In Ezio's hands, the Apple and Staff open the Vault; inside, he finds a holographic figure that identifies him as The Prophet that has brought Desmond and the 21st century observers to the Vault to listen her words. The figure, calling herself "Minerva", reveals that she and others were part of a more advanced society that lived alongside humans, though they eventually came to war. However, a natural, celestial disaster had occurred, wiping out nearly all life on the planet; the few remaining rebuilt humanity and had constructed several other temples around the world that will help them prevent the same disaster from reoccurring. As the hologram vanishes, Minerva calls Desmond by name and says the "rest is up to you". As the memory ends, Desmond wakes to find that Abstergo has discovered their hideout and, with his assimilated skills from Ezio and Altaïr, assists Lucy in dispatching their forces, led by Vidic, the man who kidnapped him, while Shaun and Rebecca pack away their equipment. As the four escape, Lucy postulates that Minerva may have been talking about the Sun's geomagnetic reversal, Desmond realizes that the writing on his wall left by Subject 16 at the begining of the game were actually warnings, and with time short, Desmond prepares to re-enter the Animus and continue searching his memories for more clues to the location of the remaining temples.