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- Robert Langdon: A professor of symbology at Harvard University and the protagonist of the novel. He is flown to CERN to help investigate the murder of Leonardo Vetra. He is described as wearing a pair of chino pants, turtleneck, and tweed jacket. His name is a tribute to John Langdon.
- Leonardo Vetra: A scientist working at CERN and a priest. He is researching on antimatter when he is murdered by the Assassin. He is also the adoptive father of Vittoria.
- Vittoria Vetra: The adopted daughter of Vetra. She, like her father, works with CERN. Her research focuses on biology and physics. The reader learns early in the novel that Vittoria worked with her father in their research of antimatter.
- Camerlengo Carlo Ventresca: The Camerlengo (Papal Chamberlain) during the conclave. He murdered the pope, who is later revealed to have been his biological father. His codename for dealing with the assassin, "Janus", was taken from the two-faced Roman god of beginnings and ends, and for whom the month of January was named.
- Cardinal Saverio Mortati: The most senior cardinal in the conclave, and the current Dean of the College of Cardinals. He was the Devil's Advocate for the late pope.
- Commander Olivetti: The commandant of the Swiss Guard. He is initially skeptical on the claims of Langdon and Vittoria until he talks with the Hassassin. He, along with other Swiss Guards, search desperately for the missing antimatter hidden somewhere at the Vatican. He is killed by the Hassassin at the church of Santa Maria della Vittoria.
- Captain Rocher: The second in command after Commander Olivetti. He is contacted by Max Kohler telling his knowledge on the real cause of the events. He is killed by Lt. Chartrand, who was under the impression that Rocher was an Illuminatus.
- Hassassin: The killer hired by Janus, the Camerlengo in disguise, to fulfill his plans. He is of Middle Eastern origin and displays his sadistic lust for women throughout the novel. He murders Leonardo Vetra, the Preferiti, and Commander Olivetti. He dies after being pushed from a balcony by Langdon at the Castel Sant'Angelo and breaking his back on a pile of cannonballs below.
- Maximilian Kohler: The director of CERN. He is feared at CERN despite his paralysis. His wheelchair contains electronic gadgets such as a computer, telephone, pager, video camera, and a gun. He contacts Langdon to help him find the killer of his friend, Leonardo Vetra. He blames the Church for his paralysis, due to his highly religious parents denying him medical care as a child, becoming a scientist as a way to rebel.
- Gunther Glick and Chinita Macri: A reporter and his photojournalist for the BBC. They are contacted by the Hassassin regarding the events happening in the Vatican. Glick has a notorious reputation as a sensationalist and conspiracy theorist journalist. Macri, meanwhile, is a veteran camera crew and a foil to Glick. They have the first-hand account on the events in the novel, from the beginning of the conclave to the election of Mortati as pope.
- Lieutenant Chartrand: A young Swiss Guard. He, together with Commander Olivetti and Capt. Rocher, search desperately for the antimatter hidden somewhere in the Vatican. He shoots and kills Captain Rocher after he is mistaken as an Illuminatus. Near the end of the novel, he is sent by the new pope to give the Illuminati Diamond as an indefinite loan to Langdon.
- Cardinal Ebner: One of the four Preferiti and a cardinal from Frankfurt, Germany. He is killed by asphyxiation, by means of putting dirt and soil into his mouth.
- Cardinal Lamassé: One of the four Preferiti and a cardinal from Paris, France. He is killed by punctures to his lungs, from which he bled to death.
- Cardinal Guidera: One of the four Preferiti and a cardinal from Barcelona, Spain. He is incinerated alive.
- Cardinal Baggia: One of the four Preferiti and a cardinal from Milan, Italy and the favorite to succeed as the new pope. He was drowned to death.