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Bellucci was represented by Elite Model Management and modelled for Dolce & Gabbana campaigns. She made her acting debut in the Italian television miniseries Vita coi figli (1991); she went on to play one of Dracula's brides in the horror film Bram Stoker's Dracula (1992) and then enrolled in acting classes. After appearing in Italian productions, she had her breakthrough role in The Apartment (1996), for which she received a César Award nomination for Most Promising Actress. Bellucci came to the attention of American audiences in Under Suspicion (2000) and gained greater international recognition as Malèna Scordia in Malèna (2000). Bellucci starred in the period horror Brotherhood of the Wolf (2001) and the comedy Asterix & Obelix: Mission Cleopatra (2002). She portrayed a rape victim in the controversial thriller Irréversible (2002), and Persephone in the 2003 science-fiction films The Matrix Reloaded and The Matrix Revolutions.

Bellucci was praised for her portrayal of Mary Magdalene in the drama The Passion of the Christ (2004). She played a prostitute in How Much Do You Love Me? (2005) and Shoot 'Em Up (2007), and acted in diverse roles in other films, including The Whistleblower (2010), The Ages of Love (2011), and The Wonders (2014). Her role in Ville-Marie (2015) earned her the Dublin Film Critics' Circle Award for Best Actress. At the age of 50, Bellucci appeared in the James Bond film Spectre (2015), becoming the oldest Bond girl in the history of the franchise. She later appeared in films such as On the Milky Road (2016), The Man Who Sold His Skin (2020), and Beetlejuice Beetlejuice (2024). On television, she has acted in Mozart in the Jungle (2016) and Call My Agent! (2018). She made her stage debut in 2019 as Maria Callas in Letters and Memoirs.

Bellucci starred alongside her second husband Vincent Cassel in on-screen partnerships that spanned ten years. She has remained involved in modelling, and worked as a brand ambassador for luxury brands such as Cartier and Dior. Some media outlets have labelled Bellucci a sex symbol. Bellucci received the knight insignias of the French Order of Arts and Letters in 2006 and of the French Legion of Honour in 2016. She represents Italy as a permanent member of the Academy of Motion Picture Arts and Sciences.