Mastitis is a postpartum disease in both humans and animals. Staphylococcus aureus (S. aureus) can induce mastitis by infection of the lactiferous ducts. There is no efficacious treatment for S. aureus-induced mastitis. Betulin has been confirmed to have multiple biological activities, including anti-inflammatory properties. The present study was to determine the anti-inflammatory effect of betulin on S. aureus-induced mastitis and to confirm the mechanism of action involved. In vivo, betulin ameliorated the histopathological changes that were induced by S. aureus. ELISA and qPCR results showed that betulin inhibited TNF-α, IL-1β and IL-6 production. Western blotting results demonstrated that betulin inhibited NF-κB phosphorylation but promoted the expression of PPAR-γ. Further investigations were performed in vitro with mouse Mammary Epithelial Cells (mMECs). The results indicated the betulin inhibited the activity of the NF-κB pathway and increased PPAR-γ expression and transcriptional activity. All of the results in the present study demonstrated that betulin played a protective anti-inflammatory role against S. aureus infection in mammary gland tissues and cells by activating PPAR-γ and inhibiting the activation of NF-κB.