Abstract: In order to investigate the intervention effect of punicalagin（PC） on the mastitis induced by Staphylococcus aureus（S. aureus）,and the mechanism of its improvement on the disease,mouse mastitis model and MAC-T cell inflammation model were established in this experiment,induced by S. aureus. The mRNA transcription levels of TNF-α,IL-1β, IL-6 and IL-10 in mouse mammary glands,and of inflammatory mediators and inflammatory cytokine tight junction genes（occludin and ZO-1） and key signaling molecules（TLR2 and nf-kappa B p65） in inflammatory pathways in MAC-T cells,were detected by RT-qPCR. The expression level of MyD88 protein in MAC-T cells was detected by Western-blot. The results showed that PC significantly inhibited the growth and the colonization of S. aureus in mouse mammary glands（P＜0.05）,and significantly reduced the mRNA transcription levels of TNF-α,IL-1β and IL-6（P＜0.05）, and signifieantly increased the mRNA transcription levels of IL-10（P＜0.05）. 80 μmol/mL PC also significantly reduced the content of inflammatory mediators and inhibited the mRNA transcription levels of inflamnatory cytokine TLR2,NF-κB p65 and the expression level of MyD88 protein in MAC-T cells（decreased by 44.13%, 45.89% and 56.62% respectively,P＜0.05）,and significantly up-regulated mRNA transcription levels of IL-10, occludin and ZO-1（P＜0.05）. The results suggested that PC has a significant preventive effect on S. aureus-induced mastitis,via the main mechanisms including the inhibition of inflammatory cascade amplification by TLR2/MyD88/NF-κB pathway and the up-regulation of tight junction protein expression to correct the dysfunction barrier of S. aureus-induced mammary epithelial MAC-T cells.