For most of the game, the player assumes the role of a young nobleman-turned-assassin named Ezio Auditore da Firenze (which when translated to English means "Eagle of Florence"). In an interview with Patrice Desilets, the game's creative director, it was revealed that Ezio's father, Giovanni, a banker and secretly an assassin, as well as both of his brothers have been executed; thus, the essence of the plot is to get revenge on the rival families that murdered them while Ezio watches over his mother and sister. Like Altaïr in the first game, Ezio is an ancestor of Desmond Miles, the modern day bartender who was captured by Abstergo, with Ezio himself being a descendant of Altaïr.
The game picks up immediately after the events of Assassin's Creed with Desmond staring at cryptic messages on his bedroom wall. Desmond has synced with his ancestors so much, a "bleeding effect" has started to form, granting him the abilities of Altaïr. He escapes Abstergo with fellow assassin Lucy Stillman. Lucy takes him to the assassins, where Desmond will use a new version of the Animus so he can train as a master assassin, by following the memories of his ancestor Ezio.
The game takes place in late 14th century and early 15th century in Italy during the Renaissance, approximately 300 years after the first game took place. Like Assassin's Creed, characters based on historical figures will be present in the game including Leonardo da Vinci, Niccolò Machiavelli, Caterina Sforza, Lorenzo de' Medici, the Pazzi Family, and Pope Alexander VI. Unlike Altaïr, whose ring finger was amputated as a sign of the Creed, Ezio's has not; his finger bears a ring displaying his family crest. This was made possible by just one of numerous technical advances made by Leonardo Da Vinci for Ezio to use.
The game starts with a young Ezio living a life of luxury and privilege in Florence, Italy. He gets into a scruff with the son of a prominent family, Pazzi, who insults his parents. Reprimanded gently by his elder brother, Frederico and his father, Ezio becomes more involved with his family's affairs. His father, Giovanni, and another man, Uberto Alberti are discussing secret matters that Ezio can't make sense of, but seem urgent. Eventually, guards raid the Auditore home and arrest Ezio's father, elder and younger brothers,Fredrico and Petruccio. Ezio delivers evidence that can prove his family's innocence to Uberto, who promises to defend them at the hearing. But it turns out Bernaldi is a traitor who destroyed the evidence and sentenced the three Firenze men to death. Swearing vengeance, Ezio has to hide his mother and sister and learn the ways of the assassin. He is first taught stealth by Paola of the Courtesans, and later combat, by his uncle Mario, of the Mercenary faction.
After assassinating Uberto, apparently a city official in charge of the law, Ezio has to balance his life between assassinations and protecting his sister and mother, who refuses to speak after Ezio's father and brothers were taken. Now wanted in Florence, Ezio has to flee Firenze to Tuscany with his family to the Auditore villa. He meets up with Uncle Mario, whose mercenaries save his family from an attack by Pazzi. Mario then takes Ezio to the villa and trains him to fight and encourages him to follow in his father's footsteps as an assassin. However, Ezio does not want to become an assassin, but rather, simply wants to leave Italy and take care of his family.
Locations included in the game include Venice, Florence, the Tuscan countryside San Gimignano and Forlì. Specific landmarks include St Mark's Basilica, the Grand Canal, the Little Canal, the Rialto Bridge, Santa Maria del Fiore, Santa Croce, Palazzo Vecchio, Ponte Vecchio, and Santa Maria Novella.