When a crooked rancher, Rance McGowan (J.P. McGowan), and his men attempt to steal a herd of valuable wild horses, it's up to the Three Mesquiteers, Stony (Robert Livingston), Tucson (Ray Corrigan) and Lullaby (Max Terhune), to see to it that they don't succeed, and that none of the horses are harmed. In order to get a law revoked which protects these wild horses, McGowan substitutes his own killer horse, Volcano, for the protected pinto stallion. He then sends the horse, and several others, off destroying crops and stampeding across the land. When Volcano kills the sheriff, the other ranchers want to repeal the law protecting the pinto stallion from being harmed. The horse is ordered to be captured. With Tucson made the new sheriff, the Three Mesquiteers go out to trap the horse.

Meanwhile, Tucson and Stony, once best friends, become increasingly estranged. First because Stony does not believe the pinto stallion is a killer, and refuses to see him put to death. The second bone of contention between them is Rita (Rita Cansino), a showgirl at the local saloon. Stony has fallen for her, but Tucson knows Rita doesn't love him. And she soon proves him right when after hearing that Stony is penniless, she calls off their marriage plans. Rita then accepts a bribe to leave town for New York and jilt Stony.

Before the pinto stallion is killed, the Three Mesquiteers learn that the real killer was McGowan's horse, Volcano, painted to look like the pinto stallion. With everything as it was before, the stallion is safe and Stony and Tucson's friendship is restored.

In Hit the Saddle, Rita Cansino plays "Rita", a femme fatale who nearly breaks up the Three Mesquiteers. She has one solo dance number, as well as a song which she sings with Robert Livingston. Released on March 3, 1937, Hit the Saddle was the second to last film she made as Rita Cansino, and the last released.