Set two years after the events of the first film, Gaylord "Greg" Focker and his fiancée Pam Byrnes decide to introduce their parents to each other. They first fly to Oyster Bay, New York, on Long Island, to pick up Pam's father, retired CIA operative Jack Byrnes, her mother Dina and one-year-old nephew Little Jack (the son of Bob and Debbie Banks). But rather than going to the airport as planned, Jack decides to drive the family to Miami to meet the Fockers in his new RV.
Once they arrive, they are greeted by Greg's eccentric but fun-loving and amiable father, Bernie, and mother, Roz, who is a sex therapist for elderly couples. Concerned that Jack might be put off by the Fockers' lifestyle, Greg convinces Roz to pretend that she is a yoga instructor for the weekend. However, small cracks begin to form between Jack and the Fockers, due to their contrasting personalities. The meet gets off to a bad start when a chase between the Fockers' sexually active chihuahua, Moses, and the Byrnes's cat, Jinx, culminates with Jinx flushing Moses down the RV's toilet, forcing Bernie to destroy it to save Moses. Later, Bernie accidentally injures Jack's back during a game of touch football.
Meanwhile, Pam informs Greg that she's pregnant, and the two decide to keep it secret for now. Jack again becomes suspicious of Greg's character when they are introduced to the Fockers' housekeeper, Isabel Villalobos. Bernie reveals that Greg lost his virginity to Isabel 15 years earlier. Jack later takes the RV to Isabel's 15-year-old son, Jorge, to fix the toilet, but is disturbed by Jorge's striking resemblance to Greg, and the fact that Jorge never met his father, and jumps to the conclusion that he might be Greg's son with Isabel. Meanwhile, Roz, Bernie and Dina realize Pam is pregnant, but promise not to tell Jack. After discovering Roz's true profession, Dina consults her on sex tips in order to seduce Jack, but none of them works.
Greg and Jack's relationship is again put to the test when Greg is left alone to babysit Little Jack, whom Jack has been raising via the Ferber method. Despite Jack's strict instructions to leave Little Jack to self-soothe, Greg is unable to stand listening to Little Jack's cries and attempts to cheer him up by hugging him, putting on an episode of Elmo's World on TV for him to watch, and acting humorously, but inadvertently teaches him the word "asshole". Disaster strikes when Greg answers a brief phone call from Roz, allowing Little Jack to be let out of the playpen (by Jinx), get ahold of the TV remote and put on Scarface, and glue his hands to a bottle of rum. After a furious argument with the Fockers and his own family (though amends are quickly made), Jack resumes his spying on Greg and sends Greg and Jorge's hair samples for a DNA test, while inviting Jorge to the Fockers' planned engagement party in the hope of getting Greg to admit he is Jorge's father. At the engagement party, Jack, who automatically assumes that Greg knew about Jorge and has deliberately been keeping him a secret from Pam, introduces Greg to Jorge. Later, when Greg denies knowing anything about Jorge, Jack still refuses to believe him and drugs him with a shot of truth serum to make him talk. While giving a toast on stage, Greg uncontrollably blurts out that Pam is pregnant and that Jorge is his son before immediately losing consciousness.
The next morning, Pam questions Greg about Jorge, and Greg promises that he knew nothing about him before the previous evening. Pam believes him, and is willing to work things out with him. Jack has reached his breaking point and demands that Pam and Dina leave the island with him. Dina refuses and reveals to everyone that Jack had drugged Greg. Everyone turns against Jack and inform him that they were all aware of Pam's pregnancy. A shocked and hurt Jack leaves with his grandson.
Bernie and Greg pursue Jack, but are soon tasered and arrested by Deputy Sheriff Vern LeFlore, for speeding and refusing to remain in their car. Meanwhile, Jack is informed that Greg is not Jorge's father (his real father turns out to be a baseball player who also resembles Greg), and attempts to defend Greg and Bernie, but LeFlore tasers and arrests him, as well. In their cell, Greg, Jack, and Bernie are released by the local judge, Ira, who is a client of Roz and a close friend of the Fockers. Before they leave, Greg asks that Jack and Bernie stop their feud. Jack admits that he made a mistake regarding Jorge and reveals his past career in the CIA to Bernie, before apologizing for his actions and making up with the two of them.
