Marmaduke (v. Owen Wilson) is a Great Dane living in rural Kansas with a cat named Carlos (v. George Lopez) 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 - where Marmaduke meets a beautiful dog named Jezebel (v. Fergie), whose boyfriend is Bosco (v. Kiefer Sutherland), a controlling and violent dog with two minions named Thunder and Lighting. Bosco intimidates Marmaduke, who does not want to fight.

Marmaduke then meets Mazie (v. Emma Stone), who develops a crush on Marmaduke. He also meets Giuseppe (v. Christopher Mintz-Plasse), a Chinese Crested dog who is afraid of everything, and Raisin, a highly-intelligent but decidedly minute Dachshund. They get together at night and crash a party thrown by Bosco for pedigrees only, 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 (the other family pet, the cat) 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 in a dog fight, which appalls the Petco executives.

He then takes Jezebel on Mazie's dream date, which the latter watches from afar. While the family is on Don's boat, Marmaduke throws a party, with most residents of the dog park coming, save Mazie, Guisseppe, 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 gave him earlier in the movie. Mazie goes to Marmaduke's house, and Carlos tells her Marmaduke never returned. She then goes looking for him. In the morning the family discover him missing, and search for him too.

Marmaduke in the meantime has met Chupadogra, a wise, elderly English Mastiff (v. 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. Mazie and the family find him at the same time, but Mazie falls into the sewers 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 goes to the dam that the sewers lead to, and finds Marmaduke in the raging waters. He jumps in after him, and begs Marmaduke to let go of the stick he's holding onto, and let the waters carry him to Phil. He reluctantly does, and is saved. Several kids got that on video, and put it on Youtube. Since it had almost seven-hundred thousand hits,he is re-hired. Phil then thinks about moving back to Kansas, but the entire family disagrees.

He and Don are thinking of ideas for commercials, having gotten Petco. They ask each other about if the dogs could talk to each other, and even dance. It shows Marmaduke, Jezebel, Mazie, Guisseppe, 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, Buster has presumably gotten a family, and all is well.