Staphylococcus aureus is an important bacterial pathogen causing bovine mastitis, but little is known about the virulence factor and the inflammatory responses in the mammary infection. Staphylococcal enterotoxin C (SEC) is the most frequent toxin produced by S. aureus, isolated from bovine mastitis. To investigate the pathogenic activity of SEC in the inflammation of the mammary gland and the immune responses in an animal model, mouse mammary glands were injected with SEC, and the clinical signs, inflammatory cell infiltration, and proinflammatory cytokine production in the mammary glands were assessed. SEC induced significant inflammatory reactions in the mammary gland, in a dose-dependent manner. SEC-injected mammary glands showed a severe inflammation with inflammatory cell infiltration and tissue damage. In addition, interleukin (IL)-1β and IL-6 production in the SEC-injected mammary glands were significantly higher than those in the PBS control glands. Furthermore, the SEC-induced inflammation and tissue damage in the mammary gland were specifically inhibited by anti-SEC antibody. These results indicated, for the first time, that SEC can directly cause inflammation, proinflammatory cytokine production, and tissue damage in mammary glands, suggesting that SEC might play an important role in the development of mastitis associated with S. aureus infection. This finding offers an opportunity to develop novel treatment strategies for reduction of mammary tissue damage in mastitis.