Mrs. Calloway is one of the cows belonging to Pearl. She makes her home on the small farm, Little Patch of Heaven, and considers herself to be the leader of the farm animals. Because of this, she clashes with the headstrong Maggie, who had arrived on the farm after being sold by her former owner. She's generally very prim and proper, but can be temperamental. One running gag throughout the film is that she flies into a rage whenever she loses her hat.
Mrs. Calloway is first seen helping Pearl with her farm chores during the "Little Patch of Heaven" sequence. She uses her hat to catch dozens of flying eggs laid by the hens, which quickly hatch into baby chicks. Later, Maggie moves into the farm and manages to endear herself to most of the local farm animals save Mrs. Calloway (who was apparently suspicious of Maggie).
When the farm animals later learn that the bank has threatened to auction off the farm if Pearl cannot come up with $750 within three days, Mrs. Calloway and Grace get talked into entering themselves with Maggie in a show at the town fair in an attempt to win prize money. Mrs. Calloway is reluctant to do so at first, but gets convinced by Pearl's newly-hatched chicks who cheep and stare at her crossly.
In town, they accidentally go to a saloon when they get scared by sounds, mistaking them for a gunfight, and make a huge mess there. After being kicked out, they meet Buck, Sheriff Sam Brown's horse, and Maggie learns that a reward is being offered for the capture of Alameda Slim, which would provide enough money to save the farm, and tries convincing her fellow cows to help her, but Mrs. Calloway disagrees. She then gets in a fight with Maggie after the latter knocks Mrs. Calloway's hat off, which lands in the mud. Sheriff Sam Brown soon breaks up the fight by tying all three bovines up to a wagon that went to a ranch. On the way there, the cows pass by Maggie's old home, which has been auctioned off to a Mr. Yancy O'Del. Mrs. Calloway and Grace learn that Alameda Slim, a notorious cattle wrestler, stole all of the cattle, and Maggie was only spared because she had been locked in the barn after winning a show.
The three cows hide at the ranch filled with longhorn steers in an attempt to catch Slim. However, they soon discover how Slim is able to steal the cows: his yodeling puts the cows into a trance and follow him. Both Mrs. Calloway and Maggie are entranced, but Grace is unaffected, due to being tone deaf, and rolls the wagon to awaken the other two. When Buck decides to capture Slim himself, the cows follow his trail in an attempt to pass him, but lose the trail in a downpour. This leads to a fallout between Maggie and Mrs. Calloway, who accuses Maggie of dragging her and Grace along for a personal vendetta.
The next morning, Mrs. Calloway and and the cows meet Lucky Jack, a jackrabbit who lost a foot to a lucky charm. After learning that Lucky Jack lost his former home, an old mine, to Alameda Slim, the cows ask the rabbit to take them to it. The cows form a plan to capture Slim. At Grace's suggestion, Mrs. Calloway and Maggie don earplugs so that Slim's yodeling does not affect them. The cows are able to capture Slim, and flee on a steam train, with Slim's accomplices and buyer in close pursuit. However, Slim is freed by Rico, a fake bounty hunter who secretly works for Slim. Alameda Slim dons his alter ego, Mr. Yancy O'Del, and plans on attending the auction of Patch of Heaven. However, Mrs. Calloway and the others use the train to follow him and are able to expose Slim's identity to the Sheriff and Pearl, who are both shocked that the auctioneer who is buying the farm is Alameda Slim, resulting in his arrest and the farm being saved by the prize money.
When Mrs. Calloway and Grace thought Maggie left the farm for good, they start to miss her and begin saying nice things about her. However, it is revealed that Maggie did not leave and decided to stay in Patch of Heaven after she heard Mrs. Calloway and Grace say nice things about her. Mrs. Calloway then gives Maggie her hat and allows her to wear it, Maggie decides to stay in Little Patch of Heaven when she hears Mrs. Calloway and Grace, who thought that Maggie had left, say nice things about her. Mrs. Calloway then gives Maggie her hat and allows her to wear it, and the three cows, along with all the other farm animals, live a happy life on Little Patch of Heaven. Mrs. Calloway and the farm animals are last seen dancing in celebration of Pearl's farm being salvaged and re-opened.
- Although Mrs. Calloway is the leader of the farm, Maggie is the overall protagonist, having bigger plans than anyone else.
- When Mrs. Calloway dives on top of Rico, the Tarzan yell can be heard.