The film opens with John (Brad Pitt) and Jane Smith (Angelina Jolie) answering questions during marriage counseling. The couple has been married for "five or six" years, but their marriage is suffering to the point that they cannot remember the last time they had sex. They tell the story of their first meeting in Bogotá, Colombia, where they met while both were secretly on the run from Colombian authorities; since the authorities were looking for tourists traveling alone after a recent heist, the two claimed to be together to avoid being questioned. They quickly fell in love and married. John later states that Jane "looked like Christmas morning" to him on the day they met.
In reality, John and Jane are both skilled assassins working for different firms, both among the best in their field. Each is concealing their true profession from their spouse. Under these cover stories, John and Jane balance their apparently mundane marriage – which both of them find after a few years to be growing dull and suffocating – with their secretive work. When both are assigned to kill DIA prisoner Benjamin "The Tank" Danz (Adam Brody) during a transfer, they encounter each other on the job and the hit ends up being botched. After their respective companies find out about the botched hit, they are assigned to discover and eliminate the other.
After a few attempts on each other's lives, fueled by a mutual sense of betrayal, their conflict culminates in an explosively violent fight in the Smith house involving both firearms, makeshift weapons and hand-to-hand combat. After a protracted, evenly-matched fight, with their house shot to shambles, they wind up with guns in each other's faces. John declines to shoot, and lays his gun down. Jane finds she cannot shoot John either, and both succumb to their love instead. The two reunite and make love for the first time in a long time.
The newly rekindled Smith partnership is quickly threatened by their employers, who have now decided to eliminate the couple. John's best friend and coworker, Eddie (Vince Vaughn), turns down a bounty of $400,000 for each Smith (since he won't get out of bed for anything less than $500,000), but John and Jane find themselves under fire from an army of assassins. Fending off an attack which blows up their house, the Smiths steal their neighbor's minivan and successfully destroy their attackers' three pursuing armored BMW sedans, all while bickering over their fighting styles and newly discovered personal secrets.
After meeting with Edd, the Smiths decide to fight together to preserve their marriage. They kidnap Danz from his high-security prison in order to give their employers something they want more than the Smiths. Danz reveals that he was merely bait, hired jointly by their employers after it was discovered that the Smiths were married, in the hopes of having one Smith kill the other. John and Jane forgot their separate contingency plans and make their stand together. In the final fight scene of the film, the Smiths – now working smoothly together as a team – defeat an extended attack by a large force of armed personnel during a long shoot-out inside a department store.
The film ends with the couple meeting the marriage counselor (William Fichtner) again, where the Smiths state how much their marriage has thrived and how happy they really are, with John motioning a subtle "10" when asked about the "sex question" once more.
It is not explained why the companies made no further attempts on the Smiths, but one can deduce that either all the assassins on the payroll were killed and the comanies found hiring new ones too expensive, or that killing the Smiths turned out to be too big a drain on corporate resources and they sent the Smiths a message to stay underground as a normal couple and they'd be left alone. The statement by Jane that they "redid the house" indicates they have resumed their former identities.