CERN director Maximilian Kohler discovers one of the facility's physicists, Leonardo Vetra, murdered. His chest is branded with an ambigram of the word "Illuminati". Kohler contacts Robert Langdon, an expert on the Illuminati, who determines that the ambigram is authentic. Kohler calls Vetra's adopted daughter Vittoria to the scene, and it is ascertained that the Illuminati have stolen a canister containing antimatter — a substance with destructive potential comparable to a nuclear weapon. When at CERN the canister is stored in a unique electrical charger which ensures the antimatter's stability but when removed its back-up battery provides power for 24 hours after which the anti-matter will self-destruct. The canister is somewhere in Vatican City, with a security camera in front of it, as its digital clock counts down to the explosion.
Langdon and Vittoria make their way to Vatican City, where the Pope has recently died. They are told that the four Preferiti, the cardinals who are most likely to be elected pope, are missing. Langdon and Vittoria search for the Preferiti in hopes that they will also find the antimatter canister. Their search is assisted by Camerlengo Carlo Ventresca (the late pope's closest aide) and the Vatican's Swiss Guard.
Langdon attempts to retrace the steps of the "Path of Illumination", a process once used by the Illuminati as a means of inducting new members; aspirants to the order were required to follow a series of subtle clues left in various landmarks in and around Rome. The clues indicate the secret meeting place of the Illuminati. Langdon sets off on the Path of Illumination in hopes of delivering the Preferiti and recovering the antimatter canister.
The Path leads Langdon to four locations in Rome, each associated with one of the primordial elements: 'Earth', 'Air', 'Fire', and 'Water'. Langdon finds one of the Preferiti murdered in a way thematically related to each location's related element. The first cardinal was branded with an Earth ambigram and had soil forced down his throat, suffocating him; the second was branded with an Air ambigram and had his lungs punctured; the third was branded with a Fire ambigram and was burned alive; and the fourth was branded with a Water ambigram and was wrapped in chains and left to drown at the bottom of a fountain.
After finding the bodies of the first two Preferiti, Langdon hurries to the Santa Maria della Vittoria Basilica and finds the Preferiti's abductor in the act of setting the third cardinal on fire. The kidnapper is an unnamed assassin who is working under the orders of the Illuminati master "Janus", whose true identity is unknown. Commander Olivetti is killed and the assassin kidnaps Vittoria. Langdon escapes and accosts the assassin at the final element's landmark (Water), but is unable to save the cardinal.
Langdon must complete the Path of Illumination in order to find the assassin and rescue Vittoria. His search leads him to Castel Sant'Angelo, which hides a tunnel leading directly into the pope's chambers in the Vatican. Langdon frees Vittoria, and together they send the assassin falling several hundred feet to his death. The two hurry back to St. Peter's Basilica, where they find that Kohler has arrived to confront the Camerlengo in private. Langdon and Vittoria fear that Kohler is Janus, and that he has come to murder the Camerlengo. Hearing the Camerlengo scream in agony from being branded with the Illuminati Diamond, the Swiss Guards burst into the room and open fire on Kohler. Just before he dies, Kohler gives Langdon a videotape that he says to show to the media.
With time running out, the Swiss Guard evacuates the Basilica. The Camerlengo rushes back in, claiming that he has received a vision from God revealing the location of the antimatter canister. With Langdon in pursuit, the Camerlengo ventures into the catacombs and finds the canister sitting atop the tomb of Saint Peter. Langdon and the Camerlengo retrieve the antimatter and get in a helicopter with only minutes to spare. The Camerlengo manages to parachute safely onto the roof of St. Peter's just as the canister explodes harmlessly in the sky. The crowd in St. Peter's Square look in awe as the Camerlengo stands triumphantly before them. Because of this "miracle", the papal conclave debate whether to elect the Camerlengo as the new Pope. Langdon manages to survive the explosion by using a window cover from the helicopter as a parachute, and lands in the Tiber River.
After viewing Kohler's tape, Langdon, Vittoria, and the cardinals confront the Camerlengo. Shortly before the beginning of the novel, the Pope met with Leonardo Vetra who believed that anti-matter was capable of establishing a link between humanity and God. Vetra's beliefs caused great discomfort to the Camerlengo. While discussing Vetra, the pope reveals that his support is due to science having given him a son. Without waiting to hear the explanation (that the child was the result of artificial insemination), and horrified that the Pope appeared to have broken his vow of chastity, the Camerlengo plots to "rectify" the situation. He poisoned the pope and, under the guise of an Illuminati master (Janus), he recruited the assassin to kill Vetra, steal the antimatter, and kidnap and murder the Preferiti. The Camerlengo planted the antimatter in St. Peter's, feigned his last-minute vision from God, and retrieved the canister just in time to save the Vatican from the ensuing explosion. This was in hope to unite the struggling Catholic Church. The Illuminati "involvement" was merely a plot engineered by the Camerlengo to cover his own plans. Upon the discovery and the Camerlengo's attempts to justify his killing of the Pope, Mortati, Dean of the College of Cardinals, reveals that Carlo Ventresca (the Camerlengo) was in fact the biological son of the late pope, conceived with a nun through artificial insemination. Overcome with guilt, Ventresca soaks himself in oil and sets himself on fire before a crowd of onlookers in St. Peter's Square. His ashes are recovered by Mortati, who places them in an urn inside his father's sarcophagus. It is revealed that the Cardinals' endorsing of him would in fact have made him Pope by acclamation. Mortati is elected his successor by the conclave, and Langdon and Vittoria reunite at Hotel Bernini where they share an extensive meal before making love. The last brand, the long-lost "Illuminati Diamond", is delivered by a Swiss Guard to Langdon on an indefinite loan, provided he would return it to the other Illuminati brands - long-since owned by the Vatican - through his final will.