Gaylord Myron "Greg" Focker (Ben Stiller) and his fiancée Pam Byrnes (Polo) decide to introduce their parents to each other. They first fly to Oyster Bay, Long Island, to pick up Pam's father, retired CIA operative Jack Byrnes (De Niro), her mother Dina (Danner) and one-year-old nephew Little Jack. 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 (Hoffman), and mother, Roz (Streisand), who is a sex therapist for elder couples. Worried that Jack may be put off by the Fockers' lifestyle, Greg convinces Roz to pretend that she is a yoga instructor for the weekend. Though Jack and Bernie get off to a good start, small cracks begin to form between Jack and the Fockers, due to their contrasting personalities. Things are made worse when a chase between the Fockers' dog, Moses, and the Byrnes' cat, Jinx, culminates with Jinx flushing Moses down the RV's toilet, forcing Bernie to destroy it to save Moses, later on Bernie accidentally injures Jack's back during a game of football.
Pam, meanwhile, informs Greg that she is pregnant, but the two decide to keep it a secret from Jack, who does not know they are having sex. Jack, however, becomes suspicious of Greg's character again when they are introduced to the Fockers' housekeeper, Isabel Villalobos (Alanna Ubach), with whom Bernie reveals Greg had a sexual affair fifteen years before. Jack later takes the RV to Isabel's fifteen-year-old son, Jorge (Ray Santiago), to fix the toilet, but is disturbed by Jorge's striking resemblance to Greg and begins to suspect he may be Greg's son with Isabel. Meanwhile, Roz, Bernie and Dina realize Pam is pregnant, but promise not to tell Jack. Growing envious of Bernie and Roz's active sex life, Dina consults Roz on sex tips in order to seduce Jack, but none of them work.
Things eventually come to a crunch when Greg is left alone to babysit Little Jack, whom Jack has been raising via the Ferber method. Despite Jack's instructions to leave Little Jack to self-soothe, Greg cannot bear to listen to Little Jack's cries and tends to the boy to cheer him up, turning the television on, acting funny and inadvertently teaching Little Jack to say "asshole". A brief phone call from Roz is long enough for Little Jack to wander out of his pen (after Jinx accidentally opens it), put on Scarface and glue his hands to a rum bottle. Meanwhile, Moses proceeds to engage in sodomy with Jinx. After a furious argument with the Fockers and his own family (though amends are quickly made), Jack reverts to his old ways and sends Greg and Jorge's hair samples for a DNA test, while inviting Jorge to the Fockers' planned engagement party in hopes of getting Greg to admit he is Jorge's father.
At the engagement party, Jack introduces Greg to Jorge, and later when Greg refuses to admit Jorge is his son, Jack injects him with a truth serum to make him talk. On stage, Greg blurts out that Pam is pregnant and that Jorge is indeed his son (in a comically Darth Vader-esque manner) before finally passing out. The next morning, Pam questions Greg about Jorge, and Greg admits that he might be Jorge's father, but insists that he knew nothing about him before the previous evening. Pam believes him, and is willing to work things out with him. Eventually Dina reveals to everyone else Jack's actions, and another argument ensues and Dina admits that they all knew Pam was pregnant and deliberately did not tell him. Shocked and hurt by this, Jack leaves with his grandson. Bernie and Greg give pursuit, but are tasered and arrested by a corrupt deputy sheriff, LeFlore (Tim Blake Nelson), for speeding. Jack returns to defend them after being informed Greg is not Jorge's father (his real father turns out to be a baseball player who also resembles Greg), but the overzealous LeFlore tasers and arrests him as well. In their cell, Greg, Jack and Bernie make up and are released by the local judge, Ira (Shelley Berman), who is a client of Roz and close friend of the Focker family.
Greg and Pam are married that weekend by Pam's ex-fiancée (Owen Wilson). During the party, Jack asks Roz for some sex tips and sneaks into the RV with Dina.
During the credits, Jack watches hidden baby-cam footage of the Fockers giving attention to Little Jack over Jack's previous objections: Roz gives Little Jack chocolate, Bernie advises him to use his crying to disagree with everything Jack says, and Greg pretends to drunkenly tell Little Jack to keep it a secret that he left to smoke pot, not answer the phone, when he left Little Jack unattended which resulted in Jack gluing his hands to a rum bottle and that Pam is not really pregnant and only said it so that Jack would let them get married. Greg then pretends to only just discover the camera but then after making mocking gestures at it, Greg reveals that he knew about it all along and none of the things he said before were true.