The game starts with Sam Fisher and his old friend Victor Coste who are about to depart from Andersen Air Force Base in Guam when an unknown enemy force destroys the entire base. Assisted by hacker specialist Charlie Cole, Sam and Vic manage to escape, although Vic is severely injured from protecting Sam from a grenade. Soon after, a terrorist organization calling itself "The Engineers" claims responsibility for the attack and announces that this attack is the first of a deadly countdown of escalating attacks on United States assets which they call "The Blacklist." They declare that they will halt the attacks only after the U.S. Government recalls all American troops deployed abroad.
In response, President of the United States Patricia Caldwell, who had just shut down the corrupt Third Echelon, assigns Fisher, Charlie Cole, Isaac Briggs and Anna 'Grim' Grímsdóttir to a newly created special operations and counter-terrorism unit called "Fourth Echelon." She installs Sam as the commander and charges the unit with the task of hunting down The Engineers and stopping them. Fourth Echelon's base of operations is a customizable cargo plane, codenamed "Paladin."
Sam and his new team's first course of action is to capture Andriy Kobin from a CIA safe house in Benghazi, Libya and obtain information from which they can learn more about The Engineers' plans. Sam successfully extracts Kobin and arrests him, keeping him aboard the Paladin. Kobin reveals some information about his business associates in Mirawa, Iraq. Infiltrating an insurgent stronghold there, Sam learns that the Blacklist attacks are more serious than originally thought. Sam arrives at a death chamber to find an executed US soldier whose execution was taped. The executioner was Majid Sadiq (Carlo Rota), a terrorist and former MI6 agent. In the video, Sadiq directly tells Sam to stop intervening or suffer the consequences.
Intelligence from Mirawa points to the next Blacklist attack being in Dallas, Texas. Sam asserts to Fourth Echelon that a direct attack would be too simple and that the true target is another city. Charlie notes that the TSA list of "do not fly" citizens was offline for roughly six hours with a group of men traveling through Chicago's O'Hare International Airport during the time the list was offline. The target is a water filtration plant off Navy Pier, which is scheduled to release a hazardous biological agent into the water supply of Chicago. Sam stops the devices and shuts off the outflow valve, sparing Chicago.
Kobin reveals to Sam back on the Paladin intelligence about one of his associates, a dealer named Reza Nouri who supplies mercenaries for hire. Sam assaults his mansion in Ciudad del Este, Paraguay and interrogates Nouri. Nouri states that to whom he lends his men is none of Sam's business. During the interrogation, Nouri's mansion comes under attack from suspected operators of Quds Force. Nouri agrees to give up Sadiq in exchange for a safe extraction.
Grim and Charlie gain intelligence about a sleeper cell of The Engineers in London, England. The cell has been activated and Sam infiltrates an abandoned mill to gain intelligence on The Engineers' next plans. Sam finds shipping documents from Iran, confirming the suspicion that Iran and Quds Force were involved in the Blacklist attacks. With the last of the chemical bombs about to leave the shipping bay, Sam sneaks onto the truck and places a GPS tracking device inside the bomb. Sam is exposed to near lethal amounts of the chemical agent and, while trying to exfiltrate, is captured alive by Sadiq. Briggs, who was providing sniper overwatch, is forced to rescue Sam, but lets Sadiq escape.
After healing from the exposure to the chemical weapons, Sam infiltrates the former US embassy in Tehran, Iran, which has been transformed into a Quds Force headquarters. Sam installs a flash drive on a mainframe computer given to him by Charlie, which is programmed to search and rip anything related to the Blacklist attacks. The flash drive reveals nothing, proving that Iran is not behind the Blacklist attacks, reversing previous suspicions.
Back on the Paladin, Briggs informs Sam that the GPS tracker placed inside the bomb in London has reached US soil in Philadelphia, Pennsylvania. Grim analyzes that there are a total of four bombs containing hazardous nerve agents. The bombs arrive at the transit yards in Philadelphia, forcing Sam and Briggs to disarm the bombs before the nerve agents are released into the mass transit system. Grim's analysis also assess that a high value Engineer target is present. After Sam and Briggs successfully defuse the fourth bomb, Sam gives chase to the Engineer leader in the transit system. The Engineer escapes the train, but is shot dead on site by responding police officers.
Returning to the Paladin empty handed, Fourth Echelon is given a break when Nouri is checked in as a prisoner in Guantanamo Bay, Cuba. Sam and Briggs form a plan to break into Guantanamo to interrogate Nouri again under Briggs' CIA credentials. Sam successfully interrogates Nouri who confirms that Sadiq is the mastermind behind the Blacklist attacks, but is unable to tell Sam when the next attack will be. Nouri also admits that Sadiq knows of Fourth Echelon and is aware of the team's individual identities. Sam evades capture and escapes the prison, riding off from the bay with the help of Briggs in a boat. Briggs drives himself and Sam to the Paladin, which has landed at an airstrip in the Yucatán province of Mexico.
Before Fourth Echelon can take off, the Paladin comes under assault by terrorists. Sam and Fourth Echelon fight off the attack, which Charlie reveals was his fault for contacting an old friend while landed. After taking off, the Paladin comes under a cyberattack directly from Sadiq. All of the intelligence on board the Paladin is compromised, as well as the on-board computers, leading the Paladin towards a crash in the Gulf of Mexico. Fourth Echelon successfully regains control of the Paladin, averting the crash. During the cyberattack, Sadiq successfully executes the "American Fuel" Blacklist attack on Sabine Pass, Louisiana, the largest oil reserve in the US.
While the US government activates continuity of government procedures, Sam assaults Sabine Pass, shutting down the leaking oil from reaching other reserve posts, averting a chain reaction. Another high value Engineer is present, to whom Sam gives chase. The Engineer reveals the true and final plans of Sadiq: to extract all US military secrets. Knowing continuity of government procedures, Sadiq successfully kidnaps a group of high level civilian and military personnel, including the Secretary Of Defense, and holds them at the Site F government bunker under Denver International Airport (DIA).
Fourth Echelon calls President Caldwell, who directly orders Fourth Echelon to divert from Denver. The Paladin pilots refuse to defy orders, so Sam orders Kobin to relieve the pilots of duty. Kobin successfully drops Briggs and Sam during a blackout landing at Denver. Briggs and Sam assault DIA, tasked with planting a virus which Charlie custom-built to safeguard against the transfer of files to Sadiq. Briggs surrenders to Sadiq in the computer terminal, separated from the other hostages. Sadiq's men torture the Secretary of Defense into complying with their demands, but Briggs intervenes, killing the Secretary before he can begin the data transfer.
The Engineers attempts to flee Site F using Briggs and the other hostages as human shields. It is revealed that Sam is disguised in an Air Force uniform at the very rear of the hostage group. Sam breaks free from his captors as Delta Force marksmen open fire on Sadiq and his men, and Sadiq escapes on foot after wounding Briggs. An unarmed Sam gives chase, disarming and assaulting Sadiq, before wounding him and taking him into custody. Even upon defeat, Sadiq claims that he has already "won," threatening Sam that if he kills him, the twelve nations who are secretly backing The Engineers will rise and if he puts him on trial, he will "spill every secret he knows." Leaving him with no choice, Sam employs the Fifth Freedom and unofficially imprisons Sadiq, while President Caldwell announces that he is killed and the game ends with Fourth Echelon prepares to continue operations. In the post-credits, it is revealed that Vic had recovered from his injuries and both he and Sam are seen about to interrogate the captive Sadiq.