The story of the game shared similarities to the story of the Assassin's Creed: Brotherhood, aside from a few exceptions:
- Lucrezia and Caterina were located in Florence, as opposed to the Castel Sant'Angelo in Rome.
- Ezio was required to kill Lucrezia Borgia in order to free Caterina Sforza, instead of capturing her.
- Claudio, a member of the Thieves Guild of Rome, was captured by the Followers of Romulus.
- The leader of the Followers was Romulus himself.
Also, the Assassin could use a hook to kill guards or to grab onto a statue and drop it on top of guards from above.
Differences from the main game
- The entirety of the game is set in 1486, instead of 1500 – 1507 in the main game despite events following similarly to the canonical version, with the story taking off from the Siege of Monteriggioni. This is, however, consistent with the mobile version of the prequel, Assassin's Creed II, which is also set entirely in 1486.
- There is no mention of Templars throughout the entire game.
- As with the mobile prequel, Ezio Auditore has a grappling hook and a halberd as part of his arsenal, though he lacks the Poison Blade, crossbow, smoke bombs, and throwing knives he wields in the main game.
- 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.
- At one point, Ezio proves capable of assassinating a guard by severing the cross at the spire of a tower, then riding it many stories below directly onto the guard, impaling him, without any injury from the fall.
- Ezio is capable of dodging bullets from pistols, in the same manner that later Assassins such as Arno Dorian, Evie Frye, and Jacob Frye do.
- Ezio Auditore uses the Flying Machine regularly as a means of transportation between cities, whereas in the console game, it only appears as a prototype in a mission to destroy its blueprints.
- Whereas the main game is set almost exclusively in Rome, the mobile version takes place in Florence and Venice as well, almost in equal measure to Rome.
- Specifically, Caterina Sforza is imprisoned by Lucrezia Borgia in Florence, where Juan Borgia is also assassinated; Octavian de Valois is found in Venice.
- During Ezio's journey from Florence to Venice on his Flying Machine, he decides that he might as well destroy the entire Borgia armada in the midst of his escape. This results in a boss battle against a giant Borgia battleship run by watermills that is never present in the main game.
- The Followers of Romulus are known as "Wolfmen" instead. Their leader is also specifically given the name Romulus. Only Romulus wears the typical wolfskin outfit of their cult while all the other Wolfmen in the game are dressed as normal guards.
- The Assassin apprentices of Rome are not largely recruited and trained by Ezio Auditore. Instead, they are presented as a group independent of him already operating in the city at the time.
- Claudio is not explicitly said to be a thief, but an ally of the Assassins nonetheless. Rather than injured in an attack against the Borgia, he is abducted by the Wolfmen. Whereas in the main game, Ezio Auditore reinforces the trust of La Volpe, Assassin leader of the Thieves faction, by taking the initiative to save Claudio, in this version, he is expressly given the task of rescuing Claudio by the Assassin apprentices to earn their allegiance.
- Caterina Sforza is captured by the Borgia not during the Siege of Monteriggioni, but after Ezio's assassination of Romulus in relatiation for it.
- A few characters' names are different:
- The confrontations and deaths of all targets are different:
- The Wolfmen are presented as the very first enemies Ezio encounters. They are destroyed with the death of their leader, Romulus, at the Colosseum in the first level when Ezio rescues Claudio. Romulus is killed while moving to engage Ezio when the Assassin kick-flips him into a rack of spears that impales him, with another Assassin slashing him with a heavy axe in the process.
- Ezio confronts Lucrezia Borgia in Florence because Caterina Sforza is imprisoned by her at the Basilica di Santa Maria Novella instead of the Castel Sant'Angelo in Rome. When Lucrezia refuses to hand over the key to Caterina's cell, Ezio kills her by shooting her throat with his Hidden Gun after choking her; an Assassin apprentice from afar follows through by firing a ballista bolt into her, pinning her against the wall.
- Juan Borgia the Elder is assassinated in Florence shortly after Lucrezia while attempting to flee on horseback; he is shot down by Ezio from afar with a ballista when the Assassin loses his horse and is unable to keep up with the chase.
- Octavien de Valois is killed in Venice while traveling on a gondola with his guards. While on his Flying Machine, Ezio uses his grappling hook to grab the baron by the neck, dragging him into the sky, then towing him into spikes protruding from a building.
- Michelotto is trapped with his guards at the Pantheon by Ezio and an Assassin apprentice. The apprentice kills all his guards, before proceeding to execute Michelotto in coordination with Ezio using halberds. In the main game, he is confronted at the Colosseum, but his life is spared by Ezio.
- The confrontation with Cesare Borgia in this version merges his two final confrontations in the main game. Cesare Borgia is met by Ezio alone at the Colosseum rather than by all the Assassin leaders at the Piazza del Popolo. Instead of being suddenly arrested by Fabio Orsini under orders by Pope Julius II, only to escape later and fight Ezio to the death years later in Viana, Cesare has his final duel with Ezio right then and there. Also unlike the main game, Ezio personally executes him in a prolonged combo and brutal fashion. He first kicks him in the face, then stabs him and slashes him multiple times with his Hidden Blade while strangling him with his other hand. After seizing back the Apple of Eden, he immediately uses it to unleash a pillar of lightning that blasts Cesare away, and, with speed augmented by the Apple, instantly dashes behind Cesare to kick him back before he lands. Once more, he speeds forward to intercept Cesare, smashing him to the ground, from where he then executes him by plunging his sword into his skull. In the console version, Ezio merely drops Cesare to his death from the wall of Viana Castle, not bothering with such a protracted demise as he does not seek his death from an insatiable lust for vengeance.
- Because Ezio does not manage to take the Apple of Eden from Cesare before fighting him, the latter exploits it to its fullest potential during his fight. It is shown granting far more powers than in the main game, including:
- empower attacks with lightning, causing them to emit lightning bolts around the user
- greatly augment the speed of the user
- create explosions of lightning from around the user
- generate cross-shaped energy grenades that detonate in electric shockwaves upon impact
- transform the user into a powered-up state capable of levitation and granting invulnerability to all damage; in this state, the user can:
- charge a ball of lightning in his hands to thrust at targets with high speed
- crucify the target in a cross generated by electricity
- Ezio's motivation for the entire story of the mobile version is pure vengeance. There is no mention of a desire to liberate Rome from Borgia oppression or even to oppose the wider goals of the Templars. Rather, his quest is to exact vengeance for the death of Mario Auditore and re-obtain the Apple of Eden. He shows no mercy to his adversaries unlike in the canonical version and even openly expresses deep-seated hatred for his targets as he kills them.