During the events of Hitman 2, professional assassin Agent 47 and his handler, Diana Burnwood, defected from the International Contract Agency (ICA) and joined forces with rogue mercenary Lucas Grey to destroy Providence: an alliance of corporate executives and industrialists collectively wielding vast global political, military, and economic influence. While 47 and Grey seek revenge on Providence for creating and exploiting them both as cloned assassins from birth, Diana is motivated by Providence's assassination of her parents, unaware that 47 carried out the killing under Providence's control. The trio captured Arthur Edwards, Providence's intermediary referred to as the Constant, who identified the three Partners leading Providence: Carl Ingram, Marcus Stuyvesant, and Alexa Carlisle. Edwards later escaped, however, and promptly seized the Partners' corporate assets for himself.

Working together, 47 and Grey eliminate Ingram and Stuyvesant in Dubai during the inauguration of the Scepter (a fictitious representation of the Burj Khalifa), and follow Carlisle to her ancestral manor in Dartmoor to assassinate her. Following the Partners' deaths, Edwards assumes full control of Providence and deploys mercenaries who capture Grey and Diana. Grey commits suicide to ensure 47 is not captured.

47 arranges to meet hacker Olivia Hall, Grey's only other trusted ally, in Berlin. Discovering that the ICA is tailing them, 47 staves off the pursuit by infiltrating a nearby nightclub run by an outlaw motorcycle club, and eliminating several plainclothes ICA agents. Unable to locate Edwards and still on the run, 47 and Hall decide to conclusively stop the ICA by exposing it to the public. 47 eliminates the overseers of the ICA data storage facility in Chongqing, China, allowing Hall to steal and publish all of the ICA's operational data while deleting all records of 47 and Diana. The ICA is irrevocably compromised and forced to shut down, ending 47's career as a professional assassin.

Meanwhile, Edwards attempts to convert Diana to succeeding him as Constant and seeks her betrayal of 47 by finally revealing his killing of her parents. Diana seemingly double-deals both sides, accepting Edwards's offer while inviting 47 to a gathering of Providence members in Mendoza, Argentina, where he is instructed to eliminate the only people opposed to Diana's succession—Tamara Vidal and Don Yates—so that she can dismantle the organization upon assuming control. While he follows her instructions, 47's longtime trust in Diana is finally shaken when she incapacitates him with poison and confronts him with the murder of her parents.

In a nightmare, a vision of Grey persuades 47 that Diana has not betrayed him, but rather helped to put Edwards within his reach. 47 wakes up imprisoned on a train travelling through the Carpathian Mountains, with Edwards also on board. Though Edwards intends to make 47 into an assassin for Providence once more by wiping his memory with a serum injection, 47 breaks free, either kills Edwards or injects him with the serum, and flees into the nearby forest. Meanwhile, Diana assumes power as Constant and forces Providence's members to resign their leadership positions at major global corporations, destroying Providence's power structure. One year later, 47 reunites with Diana, and the pair return to their former roles as assassin and handler, respectively, as a means of keeping the power of global elites in check.

In a secret ending, if 47 injects himself with the serum while confronting Edwards, he passes out, and later awakens in a padded room, greeted by Edwards' voice telling him, "Wake up. Wake up, my friend. It's the dawn of a new day, and you have things to do"—akin to the opening of Hitman: Codename 47.