When the farmer is seen with what appears to be a barbecue grill, they become afraid that the farmer plans to eat the animals, so they escape the farm on a hot air balloon. They ended up landing in a golf course and mistaken it as their new paradise.

Otis and Pip pretend to be superheroes Cowman and Ratboy, but the other animals are not fond of their antics. Fortunately, when Otis and Pip rescue the Pizza Guys from falling off a cliff, the animals decide to join them as superheroes.

A space test monkey named Bingo crash-lands in the barnyard. The animals decide to let him stay in the barnyard, but Bingo is unsatisfied with the fact that Otis is the leader, so he tries to get rid of Otis so he can become leader.

With Peck nowhere to be seen and Freddy seen lying on Peck's feathers, the animals conclude that Freddy ate Peck and plan to ban Freddy from the barnyard (Duke locks Freddy in a 'scented' pet carrier to attract the pet store truck driver so Freddy would be adopted). Otis is determined to prove that Freddy is innocent, however.

A hedgehog named Don Bling convinces Otis to fight in a wrestling match but it turns out that all of the matches are fixed so then Otis' opponents all lose intentionally. Now, Abby, Pig and Pip must try to stop the hedgehog before Otis fights someone who is really trying.

After Mrs. Beady videotapes Otis and the gang walking and talking, she calls a news station to prove she isn't crazy with proof from the tape. The animals don't like it at all, though. Now, they try to interrupt the broadcast to not let any person see their everyday anthropomorphic behavior (and fulfill their dreams of becoming famous television stars). But it backfired, when the cameraman got a call from the network saying they're not on the air, leading the news station to change the animals' tv show back to the broadcast. So now the animals must now steal the tape to keep anyone from seeing their antics.

Otis' negligence causes Pip to get captured by Snotty Boy, who plans on feeding him to his new pet snake (who talks exactly like him).

Abby discovers Otis' secret club, but the animals don't want a female member there so they pretend the club is very boring so Abby would leave. Soon, though, Abby discovers that Otis is faking, so she creates a club herself. Now everybody wants to join her club and Otis is left alone.

Otis loses the saloon to Chubs Malone, head of the gopher underground, in a game of Fizzbin. Now, the gophers dominate the barn at night.

Bigfoot is sighted in the woods, and Otis decides to go hunting for him, but Abby finds him and brings him to the barnyard for his protection. Mrs. Beady, however, is out to capture Bigfoot and prove she is not crazy.

Otis becomes the county's new crop dusting ace. An accident causes him to quit, however, and at the wrong moment: a large swarm of locusts is about to destroy the crops.

When Otis as Cowman is assigned the task of guarding the Jurassic Corn Kernel at the county fair, Mertin Fargleman (voiced by Tom Kenny) wants to steal it to win the Gardening Award posing as Professor Twineyvines. When the town is impressed with Twineyvines and unimpressed with Cowman trying to save the Kernel from him, Twineyvines becomes the new protector. Immediately, Otis takes the kernel to protect it and is branded a thief and then chased by a mob. He then decides to stop Twineyvines when Abby finds Otis and Pip headed for Peru and says that Peck, Pig and Freddy have been kidnapped by Mrs. Beady, who they do not know is in line with Twineyvines. Now, Otis, Pip, Abby, Pig, Freddy and Peck must unite to defeat Fargleman, clear Cowman's name, and save the Jurassic Corn Kernel (mutated into a giant monster) from destroying the fair (even though the fair was already destroyed).

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Pig returns to his birthplace to be married, but after Otis realizes that Pig's bride, Brunhilda, is cruel and bratty, he tries to stop the wedding by beating Pig in a contest.

After the animals break the farmer's classic video game system, they need to make some quick cash to replace it. Seeing an ad to babysit, they dress up as British nannies and show up at the address on the ad. Only when Mrs. Beady answers the door do they realize they're babysitting Snotty Boy, but Snotty Boy wreaks havoc on them and the animals have to get the little terror fed, bathed and into bed before the Beadys get home and earn the money for babysitting.

Abby is excited that her cousin, Veronica, is coming to the barnyard for a visit, but it turns out Veronica, despite appearing to be nice on the outside, is actually using her beauty to manipulate the animals into doing things to please her.

In order to get out of going to a "Brianna Texicanna" concert with Abby so he and his friends can go bowling to beat a rival farm, Otis lies and says he needs to celebrate "Happy Animal Fun Time Day" in which everybody must stay home to celebrate. But Abby believes him and decides to cancel going to the concert and stay home to celebrate too. So Otis distracts her with the holiday's activities so that he can join with his friends at bowling and Abby doesn't see that he lied to her.

When responsibility is thrust upon Otis to watch the three kids of the barnyard, Joey the calf, Macy the lamb, and Boyle the chick, after Abby and Bessy read story time, Otis loses them upon taking them around the big city when Otis and the gang had to remove a chip from Freddy's skin to keep Abby and Bessy from finding out where they are. So Otis and the gang stage a fake robbery to reunite with the kids after a cop finds them and about to take them to the station. But soon the kids call Abby & Bessy to pick them up. On the way back to the barnyard in a helicopter, Bessy tells them about Otis being a lair and after each line, she punches Otis and with him asking for a parachute to bail the helicopter.

The animals buy a security system called The Barn Buddy (voiced by Gilbert Gottfried) for the barnyard, but it turns out that the system is doing more harm than good.

The animals liberate a show whale from a water show at a fair, but Otis has a lot more to go when the animals are being constantly eaten by this whale.

The animals get arrested when they are mistakened by the police for bandits disguised in animal costumes. Chained together, they try to escape so that they can use a computer to find the real thieves. But when they do catch the real thieves, the police says due to budget cuts, they aren't any keys to free them from their chain, so now they're stuck chained together

Otis convinces his college buddies Donner and Blitzen to stop by the barnyard on Christmas Eve. During the visit, Otis accidentally gives Santa a cup belonging to Freddy, who is sick. When Santa comes down with Ferret Fever and can't finish his rounds in the county, which turns out to be his last stop on his Christmas Eve rounds, then Otis and the animals vow to make toys and deliver them by midnight to save Christmas. Meanwhile, Snotty Boy, not knowing he’s on the naughty list, really wants a Red Rider BB Taser that Santa doesn't bring him every year. So he sets a trap for Santa to make sure he brought his present.

After Freddy gets struck by lightning, he becomes a genius and knows everything, so they get him to compete in a trivia show so they can win a million dollars. They realize that, unfortunately, Freddy's intelligence is only temporary and he becomes stupid again after a prolonged period of time. So the animals make an electrical system to make Freddy smart with each shock. But the problem is, every time a question is announced, he keeps getting dumb again and again. Frustrated that he keeps getting dumb, Freddy goes backstage and shocks himself at high velocity and dreams that he meets with Albert Einstein who gives him the answer to the final question which is actually incorrect so Freddy curses Albert Einstein for making him lose.

Otis' old best friend, Eddie comes to visit. Otis is mad because Eddie stole his product idea for pizza flavored gum. Eddie claims to have seen the error of his ways and wants new product ideas. Eddie starts hanging out with the gang and wins them over, except for Pip, who is suspicious. Pip then finds out that Eddie is just a sleazy, backstabbing jerk and reveals this to the gang. The gang decides to get rid of him by having the crows send Eddie flying off in a car made from cobs of corn from the field. Unfortunely, Eddie manages to make a bundle off the car, leaving the others in shock, except for Pig, who stupidly thinks Eddie won.

An ancient cave cow comes to attack the barn animals! Meanwhile, scientists kidnap Otis, thinking he's really Cave Cow.

Pig's mole subjects (from when he was Emperor of the Mole People for 45 minutes) have sought him out to return as their Emperor and defend them from Eric the Terrible, a verbally abusive worm and with the mole people around the barnyard, they can't get Pig to hang out with them because they keep shocking them when they are near Pig.

In the series finale, Otis orders a satellite dish online in order to watch monkey boxing from Rio. Mrs. Beady sees this and believes that he is secretly signaling Aliens. Otis finds out and tries to prank Mrs. Beady with a fake alien invasion broadcast. This broadcast is sent to real aliens who are attracted to Earth and plan to conquer and destroy the planet. The animals must stop and stall them while they figure out their weakness.