Lucy returns and breaks Desmond out of Abstergo, taking him to an Assassin safehouse and introduced to Shaun, a historian, and Rebecca, their technical support. Using an improved version of the Animus, Desmond takes witness to Ezio Auditore da Firenze, a young nobleman from the late 15th Century in Florence, shortly before the execution of Ezio's father and brothers by order of a corrupt official working for the Templars. He and his mother and sister take to hiding at the Monteriggioni villa owned by his uncle Mario, who helps to train Ezio in the ways of an Assassin. Ezio and Mario discover that Rodrigo Borgia is the figurehead of a number of Templars all who were accomplices in the execution of his family, and with help of allies like Leonardo da Vinci, methodically assassinate the lower-ranked Templars, while learning that Borgia has acquired a Piece of Eden called the Apple. Eventually, Ezio corners Borgia, who hands over the Piece and flees; Ezio is inducted into the ranks of the Brotherhood and believed to be a prophet based on their codex. A decade later, the Brotherhood learns that Borgia has since become Pope Alexander VI, using the Church's influence to strengthen the Templars. Ezio invades Vatican City and confronts Borgia, discovering that the Papal staff is also a Piece of Eden. Defeating Borgia but sparing his life, Ezio uses both the Apple and the Papal staff to reveal a chamber of ancient technology. Inside, he is addressed by a hologram of a humanoid female that calls herself Minerva who speaks directly to Desmond through Ezio. Minerva explains how her society had pre-dated humanity's, but a great disaster nearly wiped them out, and warns that another event is due to occur soon, putting the fate of humanity in Desmond's hands. At this discovery, Desmond and his allies find the safehouse has been compromised by Abstergo and flee.
Similar to the first game, Assassin's Creed takes place in historical recreations of Venice, Florence, Forlì, San Gimignano, and the Tuscan countryside, and incorporates events during that period as part of the story. Missions are divided into main story missions, themselves divided into memory sequences reflected points along Ezio's life, and side missions which can be accomplished at any time; this approach to mission structure remains consistent in the other games in the series. The Monteriggioni villa provides several functions which can be expanded on by paying money for upgrades of surrounding buildings, or by purchasing artwork, weapons, and costumes for the villa; in turn, the villa will generate wealth for the player at a rate influenced by the upgrades and acquisition of these items. Additional quests involve locating secret Assassin seals, and finding hidden marks left by "Subject 16", a former Animus user, that hint at the nature of Minerva's society.