Abstract: Staphylococcus aureus is a contagious, mastitis-causing pathogen that is highly adapted to survive in mammary gland. This study focuses on γδ T cell populations in mammary gland involved in the immune response against S. aureus. The C57BL/6J mice in 10-15th day of lactation were intramammary challenge with S. aureus isolated from goat chronic mastitis or with PBS (control). γδ T1(interferon (IFN)-γ-producing) cells and γδ T17 (interleukin (IL)-17-producing) cells in the mammary gland during infection were evaluated by cytometry. The results showed that the percentage of γδ T1 in γδ T cells decreased following intramammary injection and significance was observed at 8, 36, 48, 60 and 72 h compared with control. At the same time, expression of IFN-γ mRNA was decreased. However, percentage of γδ T17 in γδ T cells was increased significantly at 18, 48 and 60 h post challenge, accompanied by an increase in the expression folds of IL-17, IL-23p19 and CCR6 mRNA following intramammary injection. Overall, these data suggest that γδ T17 cells, not γδ T1 cells, in mammary gland tissue were activated rapidly and played an important role in mediating mammary inflammation caused by S. aureus.