- Acting for Two: Alex Ivanovici voices Lorenzo de' Medici, Bartolomeo d'Alviano and Girolamo Savonarola in English.
- Cross-Regional Voice Acting: This game, like the rest of the series, uses a mix of Montreal and Los Angeles voice actors, accompanied by Italian actors.
- Fake Nationality: While a good chunk of the cast are actually Italians, some were certainly not from the country or have Italian ancestry.
- A good example is the decidedly non-Italian and American Roger Craig Smith, the voice actor of Ezio Auditore.
- Ditto the Egyptian-born (of Lebanese descent) French-speaking Canadian Manuel Tadros as the Spanish-born Italian Rodrigo Borgia.
- Multiple Languages, Same Voice Actor: Manuel Tadros voices Rodrigo Borgia/Pope Alexander VI in both English and French.
- Playing Against Type: In the Japanese version, Ayako Kawasumi and Kaori Nazuka are normally typecasted in Anime voicing heroic or kinder roles. In this game, both actresses voiced two of the most cruelest women of the Renaissance: Lucrezia Borgia and Caterina Sforza, respectively.
- What Could Have Been: Rosa would've made future appearances in the franchise had her voice actress, Lita Tresierra, not been tragically killed in a car accident.
- You Sound Familiar: In the French version, Marc Alfos voiced both Mario Auditore and Richard the Lionheart in the first game.
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