Maggie is the only cow left on the Dixon Ranch after Alameda Slim (a cattle rustler capable of stealing 500 in a single night) stole all the rest of Mr. Dixon's cattle. Dixon sells Maggie to Pearl, a kind and elderly woman that runs a small farm called Patch of Heaven, with the help of Hubie, Marina, Rocko, Petra, Beanie, Timmy, Pikatwo, Bonkers and Fawn.
The local Sheriff arrives to tell Pearl that her bank is cracking down on debtors. Pearl has three days to pay the bank $750, or her farm will be sold to the highest bidder. Hearing this, Maggie convinces the other cows on the farm (Grace, a happy-go-lucky character, and Mrs. Calloway, who has had leadership gone to her head) as well as Hubie and his friends to go to town to attempt winning prize money at a fair.
While the cows are in town, a bounty hunter named Rico (who Buck, the Sheriff's horse, idolizes) drops a criminal off and collects the reward. Stating he needs a replacement horse to go after Alameda Slim while his own horse rests, he takes Buck. When Maggie finds out that the reward for capturing Slim is of exactly $750, she convinces the other cows and Hubie and his friends to try to capture him to save Patch of Heaven.
That night, they hide among a large herd of steers, when Alameda Slim appears. Before any of them can do anything, Slim begins a yodeling song which sends all the cattle (except Grace, who is tone deaf) into a trance that causes them to dance madly and follow Slim anywhere. In the chaos, Marina is swiped away by the Willies Brothers. Grace is able to bring Maggie and Mrs. Calloway back to their senses just before Slim closes the path behind him with a rockslide to stop Rico and his men from chasing him.
After realising that Marina is gone, Hubie vows not to leave the Wild West until she is found.
As Rico discusses with his men what his next move will be, Buck starts talking with Maggie, Grace, Mrs. Calloway, Hubie and his friends as old friends and miming actions. This causes Rico to believe Buck is frightened by cows, and sends Buck back to the Sheriff. Buck escapes, determined to capture Slim for himself to prove his worth. Maggie, Grace, Mrs. Calloway, Hubie and the others are left behind, but they meet a peg-legged rabbit named Lucky Jack, who leads them to the Slim's hideout mine.
At the mine, Slim reveals that he has been stealing all cattle from his former patrons. When his former patrons can't support their land anymore, he buys the land when it is auctioned off, under the guise of the respectable-looking Mr. O'Dell, using the very money he gets from selling the cattle he stole.
Slim also visits Marina in a cell and tells her that he had made a deal with Drake to capture Marina and take her to an alter where he will wait to make her his bride. Marina sings a song, wondering if things will be alright.
After arriving to Slim's hideout, Hubie, his friends and the cows capture Slim and find Marina. They run off with Slim's accomplices and buyer in pursuit on a steam train. Rico arrives. When the chase stops, Rico is revealed to work for Slim, much to Buck's shock.
Slim dons his Yancy O'Dell costume and leaves everyone to be locked in the steam train by Mr Wesley and the Willie Brothers, while he goes to attend the auction. But Buck redeems himself and goes back to save them. After defeating the bad guys Hubie, his friends and the cows arrive back at Patch Of Heaven using the train to the farm and expose Slim. Slim is arrested, and Patch of Heaven is saved by the reward money.
Buck however is saddened that Rico is gone and wonders what to do next. Hubie and his friends offer him to join them on their adventures in which he does.
After the credits, Drake is revealed standing at an alter near the railroad track. No one arrives and he pouts 'Well, this sucks!'