Marmaduke is a Great Dane living in rural Kansas with a cat named Carlos. His owner, Phil (Lee Pace), works for Bark Organic dog food. Phil is very strict, from Marmaduke's perspective.
One day, Carlos tells Marmaduke that he overheard Phil saying that they were being transferred to Orange County. They move from Kansas into their new house in California. Phil's boss, Don Twombly (William H. Macy), has the goal of getting Bark Organic into every Petco store in the country. Phil and Don meet at the dog park to discuss Phil's assignment an ad campaign to win over Petco. There, Marmaduke meets a beautiful Rough Collie named Jezebel (Fergie), whose boyfriend is Bosco (Kiefer Sutherland), a controlling and violent Beauceron with two Miniature Pinscher minions named Thunder and Lightning (Damon Wayans, Jr. and Marlon Wayans). Bosco intimidates Marmaduke, who does not want to fight.
Marmaduke then meets Mazie (Emma Stone), who develops a crush on Marmaduke. He also meets Giuseppe (Christopher Mintz-Plasse), a Chinese Crested dog who is afraid of everything, and Raisin (Steve Coogan), a highly intelligent but decidedly minute Dachshund. They get together at night and crash a pedigrees-only party thrown by Bosco, only to be scared away by Bosco. Marmaduke then asks Mazie to help him get a girl, whom she presumes is herself but is actually Jezebel.
Marmaduke has Carlos pretend to be lost in the dog park, and the two stage a fight in front of all the other dogs in order to boost Marmaduke's perceived toughness. Marmaduke then enters a dog surfing contest put together as a promotional stunt by Phil to sway Petco and beats Bosco, who is an established dog-surfing champion. They get into a fight, which appalls the Petco executives.
He then takes Jezebel on Mazie's dream date, which the latter watches from afar. While the Winslow family are on Don's boat, Marmaduke throws a party, with most residents of the dog park attending save Mazie, Giuseppe and Raisin. Bosco crashes the party and discovers it was Carlos at the dog park. He then exposes Marmaduke, who loses his pedigree friends. He is left with no friends and a destroyed house. When Phil discovers the house in a wreck, he locks Marmaduke outside for the night. Marmaduke runs away, and leaves Mazie a toy that she had given him earlier. Mazie goes to Marmaduke's house, and Carlos tells her Marmaduke never returned. She then goes looking for him. Marmaduke in the meantime has met Chupadogra, a wise, elderly English Mastiff (Sam Elliott) who is feared throughout Orange County for presumably killing his owner. In reality, he ran away to lead the pack, but they abandoned him. He has spent the time alone in the woods with nothing but a blanket and his old water bowl, which reads "Buster". Buster/Chupadogra tells Marmaduke to go home and return to his family, and then distracts a dog catcher. Marmaduke leaves, but gets lost.
In the morning, the family discover him missing and begin searching for him. Mazie and the family find him at the same time on the streets, but Mazie falls into the subterranean rainwater conduit after the street below her collapses. Marmaduke jumps in after her and Phil tries to retrieve him, but the fire department tries instead. The fireman saves Mazie, but loses Marmaduke in the raging water. By this time, Phil has been fired for missing the meeting for the last chance with Petco. Phil then runs to the aqueduct that the conduits lead to and finds Marmaduke in the raging waters. He jumps in after him and begs Marmaduke to let go of the branch he's holding onto and let the waters carry him to Phil. He reluctantly does, and is saved. Several kids get that on video and put it on YouTube. Since it generates almost 700,000 hits, Phil is rehired. Phil then talks about moving back to Kansas, but the entire family wants to stay in California. Marmaduke later confronts the pedigrees, saying that differences shouldn't matter, that they're all dogs and should have an equal share of the park. Everyone agrees and turn on Bosco, who leaves, vowing revenge. Meanwhile, a Youtube video of Phil rescuing Marmaduke wins the company the Petco deal and he gets his job back. He and Don are thinking of ideas for commercials. They ask each other about if the dogs could talk to each other, or even dance.
The finale then shows Marmaduke, Jezebel, Mazie, Giuseppe, Raisin, and Buster, among others, dancing and singing "What I Like About You", which turns out to be the commercial. In the end, Marmaduke and Mazie are dating, Marmaduke and Jezebel are friends and all is well.