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Background:Tea tree oil (TTO) plays an important role in antibacterial activity and alleviating the inflammatory responses. Bovine mammary epithelium and polymorphonuclear leukocytes (PMNL) can actively respond to bovine mastitis infection. However, regulatory effects of TTO extracts on the innate immune response of bovine mammary epithelial cells (BMECs) and PMNL remain not reported. Therefore, aim of the study was to evaluate the effects of TTO extracts on the mRNA levels of the genes involved in the innate immune response of BMECs and PMNL. Results:Our results demonstrated that addition of 0.025％and 0.05％TTO increased the proliferation of BMECs, and significantly enhanced (P<0.05) the viability of BMECs exposed to Staphylococcus aureus (S. aureus). An inhibitory effect was observed against the growth of S. aureus by TTO incubation. The 0.05％TTO reduced S. aureus biofilm formation, association and invasion of S. aureus to BMECs, and changed the morphological and structural features of S. aureus. The proinflammatory cytokines IL-1β, IL-6, and TNF-αwere decreased (P<0.001) by the incubation of TTO. Interestingly, the expression of IL-8 known for PMNL chemotactic function was elevated (P<0.05) by 0.05％TTO treatment. Consistently, 0.05％TTO increased the migration of PMNL in S. aureus-exposed BMECs when compared with S. aureus treatment alone (P<0.05). In addition, PMNL incubated with 0.05％TTO decreased the levels of NFKB inhibitor alpha (NFKBIA) and TNF-α. Conclusions:Our results indicate that use of TTO can relieve the BMECs pro-inflammatory response caused by S. aureus and promote the migration of PMNL to mount the innate immune responses, and it may be novel strategy for the treatment of bovine mastitis caused by S. aureus.