The game follows the main version somewhat accurately, though various differences are also visible. Set in the Italian Renaissance, Assassin's Creed II follows the story of young Ezio Auditore da Firenze as he embarks on his mission. Ezio's family had been executed via a conspiracy by the Knights Templar, and after Ezio realises his bloodline traces to that of an ancient Assassin sect, he begins his journey to avenge his family and kill the conspirators responsible.
With the help of his friend, the legendary Leonardo da Vinci, Ezio starts to assassinate his targets, beginning from Uberto Alberti, the judge who sentenced his family to death. After making his escape, Ezio then proceeds to rescue Lorenzo de' Medici from captivity, in order to find out the location of his next target, Francesco de' Pazzi, and then from there to Emilio Barbarigo. Using Leonardo's Flying Machine, Ezio goes on to assassinate Carlo Grimaldi from overhead before making his way to Sylvio Barbarigo.
Finally, Ezio faces off against the man behind the plot to kill his family: Rodrigo Borgia, the Pope. After a long fight, Ezio finally overcomes him and takes the mysterious artifact he had held, the elusive Piece of Eden. To Ezio's surprise, however, a figure appears from the Piece of Eden with a message for someone referred to as "Desmond."
Assassin's Creed II spans nine levels, scattered throughout the three cities of Florence, Venice, and Forlì, including two sections in which Ezio uses the Flying Machine to either escape or attack. Throughout the game, Ezio can use various abilities and items, ranging from running, jumping, crawling, and hanging onto ledges, to attacking with his Hidden Blade, sword, arquebus, grappling hook, boomerang, or spears (which he can salvage from defeated guards). Unlike in its mobile predecessor, Ezio can now perform a variety of new moves, including assassinating from a hanging position, and using Courtesans to distract guards and targets.
Differences from main game
- The entirety of the game, from Uberto Alberti's assassination to the climactic fight with Rodrigo Borgia is set in 1486, whereas in the canonical game, these events span two decades of Ezio's life, from 1476 to 1499.
- While Ezio does not have throwing knives or a Poison Blade, he is equipped with a boomerang and a grappling hook, both of which do not appear in the console game. He also wields a long weapon as part of his arsenal, using it both in the assassination of Emilio Barbarigo as well as to aid in his freerunning. The pole-arm is called a spear in the game but resembles a glaive.
- Ezio is capable of using the grappling hook to hang enemies from trees in an assassination technique identical to that employed by Ratonhnhaké:ton and Aveline de Grandpré.
- While hiding in haystack carts, Ezio can actually move the carts from inside, all while maintaining his cover, a function not possible in the main games.
- The sword Ezio wields is capable of deflecting fireballs, a technique he employs heavily in his final duel against Rodrigo to defeat him.
- War Machines are more prevalent in the game, with the flying machine even used by the guards to bomb Ezio as he escapes from his assassination of Uberto Alberti. Ezio himself uses it first to escape during this assassination. Like the game, he repeats its use later to kill Carlo Grimaldi, though this target himself pilots an armored war boat similar to that of Leonardo's naval cannon. Various enemies also use weaker versions of his war boat.
- Rather than Rome, the final stages of the game is set in Forlì.
- Forlì is shown not as a city, but a dark wetland—as it is in the main game—with nothing but giant, forlorn ruins.
- It is implied that the Apple of Eden is obtained by Rodrigo Borgia at an expedition to Forlì which Ezio tracks him to.
- By Rodrigo's dialogue, it is also suggested that Ezio Auditore was not aware that Rodrigo was the mastermind behind his family's execution until their confrontation.
- The relation between the characters differ:
- Leonardo da Vinci serves as Ezio Auditore's main guide for his missions, effectively being the one to issue him commands, whereas in canon, he is merely an ally of the Assassins.
- Ezio Auditore rescues Lorenzo de' Medici, but it is suggested that their relation is such that he only does so because Lorenzo knows the whereabouts of Ezio's target, Francesco de' Pazzi.
- Francesco de' Pazzi is described as a direct subordinate to Emilio Barbarigo, in turn a direct subordinate to Carlo Grimaldi.
- Several characters' names differ:
- Lorenzo de' Medici is called "Lorenzo Medici", omitting the possessive.
- Francesco de' Pazzi's name is spelled "Francesco de Pazzi".
- Silvio Barbarigo's name is spelled "Sylvio Barbarigo".
- Only several of the assassination targets in the main game are present: Uberto Alberti, Francesco de' Pazzi, Emilio Barbarigo, Carlo Grimaldi, Silvio Barbarigo, and Rodrigo Borgia. The rest are entirely absent.
- The entire Pazzi conspiracy is omitted, with the extent of it being Francesco de' Pazzi's kidnapping of Lorenzo de' Medici during a ball in Florence in 1486, under direct orders by Emilio Barbarigo himself.
- The confrontations and deaths of all targets are different:
- Rather than being assassinated by a wrathful, inexperienced Ezio Auditore in the days following the execution of the Auditore family, Uberto Alberti is instead only hunted down in 1486, years after the execution. Ezio is explicitly assigned the mission by Leonardo da Vinci himself, though vengeance is still stated to be the primary motive. Instead of being killed in Florence, Uberto is killed in Venice while traveling on a gondola with an escort, falling victim to an air assassination by Ezio.
- Francesco de' Pazzi is assassinated by Ezio while being distracted by a courtesan at a ball in Florence; the Assassin approaches him by blending in with the crowd.
- Emilio Barbargio is slain by Ezio while exiting a church, with the Assassin leaping out from a bush to kill him with a glaive.
- Carlo Grimaldi is killed in a battle of War Machines against Ezio, using his armored war boat—reminiscent of Leonardo's naval cannon—against Ezio's flying machine. The bombs from the flying machine destroy Carlo's war boat, killing him.
- Sylvio Barbarigo is assassinated by Ezio in Forlì in the midst of trying to enter a secret tomb—presumably containing the Apple of Eden.
- Rodrigo Borgia engages in a fierce battle against Ezio using his Staff of Eden as in the main game, but the powers of the Staff are markedly different. While Ezio ultimately spares him in the canonical version, in this mobile adaptation, he kills him in the battle, interrupting a last resort, ultimate technique by Rodrigo by activating a mechanism that collapses two spiked columns on top of him. Upon dying, Rodrigo's body dissolves, with wailing dark spirits fleeing from his corpse.
- The Staff of Eden is depicted as a black staff with a blue, magical orb set on top, similar to stereotypical magic staves of wizards.
- The Staff of Eden's powers differ. Among its abilities shown in the final battle, it is capable of empowering the user with a burst of speed that leaves afterimages, summoning pillars of fire from the ground, summoning boulders to drop from the sky, healing the user, generating a shield of black electricity and orbiting balls of energy, and shooting and deflecting volleys of fireballs.
- The appearances of all the targets are different, with all of them wearing identical purple robes, with Emilio and Sylvio Barbarigo donning matching hoods. Franesco de' Pazzi sports white hair and beard, which, alongside his purple cap, makes him similar in appearance to Carlo Grimaldi in the main game. Carlo himself is never seen as he is inside an armored war boat for his entire appearance.
- Although wearing similar clothes, Rodrigo Borgia has a notably different face in the mobile game and is hunchbacked, a defect that his character does not have in the canonical game.
- In the ending of the mobile version, upon killing Rodrigo, the narration in the epilogue describes a similar, though slightly different, ending to the main game. Ezio believes his quest as an Assassin to be over and desires to live the rest of his life in peace. However, the Apple of Eden immediately emits a hologram of Minerva—not identified in the game—which through him, warns Desmond Miles of the imminent destruction of humanity. This scenario does not take place within the Vatican Vault but merely occurs the moment Ezio interacts with the Apple after slaying Rodrigo in Forlì.
- In the mission taking place at the ball in Florence, the guards with firearms are described as wielding muskets rather than arquebuses. This is anachronistic as muskets were not in use until beginning in the next century. Another anachronism is the prevalence of powdered wigs at the ball, worn by nearly all the male guests, which would not be common fashion until later centuries.