Abstract: Staphylococcus aureus is one of the major pathogen which can cause mastitis in dairy animal. CD4+ lymphocytes play a significant role in clearing this type of bacteria. To our knowledge, CD4+ lymphocytes change in mouse model of S. aureus mastitis is poorly understood. To investigate the response of CD4+ lymphocytes in mammary gland, the mice mastitis was induced with S. aureus in this study. New protocol was used to separate lymphocytes in mammary gland. The results showed that CD4+ lymphocytes increased significantly comparing with blank and saline mice (p = 0.0019, p<0.01) at 72 h post Intramammary Inoculation (IMI). At the same time, the infection mice mammary gland tissues were swelled and were suffering from hyperemia. From histological observation, massive inflammatory cells are infiltrated in the tissue. The results suggest that CD4+ lymphocyte is one of important cells in against S. aureus infection in mammary gland.