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, to pick up Pam's father, retired CIA operative Jack Byrnes, her mother Dina, and Pam's infant nephew, Jack “Little Jack” Banks. Rather than going to the airport as planned, Jack decides to drive the family to Miami to meet Greg's parents in his new RV.
Once Greg, Pam, and Pam’s parents arrive in Miami, they are greeted by Greg's eccentric but fun-loving and amiable father, Bernie Focker, a lawyer-turned-stay-at-home-dad, and Greg's mother, Roz, who is a sex therapist for elderly couples. Soon, small cracks begin to form between Jack and the Fockers, due to their contrasting personalities. The gathering gets off to a bad start when a chase between the Fockers' dog, Moses, and the Byrneses' 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.
Pam informs Greg that she is pregnant, and the two decide to keep it a secret until they are married. Jack again becomes suspicious of Greg's character when they are introduced to the Fockers' former housekeeper, Isabel Villalobos. Bernie reveals that Greg lost his virginity to Isabel 15 years earlier. Isabel's 15-year-old son Jorge, who has never met his father and bears a striking resemblance to Greg, catches the attention of Jack. Meanwhile, Roz, Bernie and Dina realize Pam is pregnant, but promise not to tell Jack.
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 and acting humorously, but inadvertently teaches him the word "asshole". Greg answers a brief phone call from Roz, and Little Jack is let out of the playpen by Jinx and he glues his hands to a bottle of rum. Jack resumes his spying on Greg and sends Greg and Jorge's hair samples for a DNA test, while inviting Jorge to Greg and Pam's engagement party in hopes of getting Greg to admit he is Jorge's father. At the engagement party, Jack, who assumes that Greg knows about Jorge and has deliberately been keeping him a secret from Pam, introduces him 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. While giving a toast, 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 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 tasered and arrested by Officer Vern LeFlore for speeding and refusing to remain in Bernie’s car when pulled over. Meanwhile, Jack receives the results of the DNA test that determines Jorge's father is really a baseball player who also resembles Greg. When Jack sees Bernie and Greg being pulled over, he attempts to defend them, but LeFlore tasers and arrests him as well. In their cell, Greg, Jack, and Bernie are released by Judge Ira, a client of Roz. 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. Greg and Pam are married that weekend by Pam's former fiancé, Kevin, who is now an ordained interfaith minister.
Robert De Niro as Jack Byrnes
Ben Stiller as Greg Focker
Dustin Hoffman as Bernie Focker
Barbra Streisand as Roz Focker
Blythe Danner as Dina Byrnes
Teri Polo as Pam Byrnes
Owen Wilson as Kevin Rawley
Spencer Pickren and Bradley Pickren as Jack “Little Jack” Banks
Alanna Ubach as Isabel Villalobos
Ray Santiago as Jorge Villalobos
Tim Blake Nelson as Officer Vern LeFlore
Shelley Berman as Judge Ira
Cedric Yarbrough as Prison guard
followed by a sequel, Little Fockers, in 2010.