Knowledge in the effects of oxidative stress and antioxidants on bovine mammary epithelial cells (bMEC) is limited. The objectives of this study were to investigate the oxidative damaging effects of hydrogen peroxide (H₂O₂) and the cytoprotective effects of resveratrol, a well-known natural product rich in grape seeds, against oxidative stress in cultured bMEC (MAC-T). To establish an oxidative stress model in bMECs, 500 μM H₂O₂ was added to MAC-T cells. The CCK-8 assay was applied to detect cell viability and flow cytometry method was used to detect intracellular production of reactive oxygen species. Pretreatment of MAC-T cells with resveratrol could rescue the decrease in cell viability and resulted in lower intracellular accumulation of reactive oxygen species after H₂O₂ exposure. Using qRT-PCR, we found that resveratrol helped MAC-T cells to prevent H₂O₂-induced endoplasmic reticulum stress, indicated by significantly decreased abundance of endoplasmic reticulum stress marker GRP78 and CHOP mRNA (P < 0.01). Resveratrol also inhibited mitochondria-related cell apoptosis by downregulating the expression of pro-apoptotic Bax gene (P < 0.01) and upregulating expression of anti-apoptotic Bcl-2 gene (P < 0.01) compared with H₂O₂ group. Moreover, resveratrol increased the abundance of multiple antioxidant defense genes (HO-1, xCT, Txnrd1, and NQO-1) in MAC-T cells under normal/oxidative conditions. It is confirmed that Nrf2 was required for the cytoprotective effects on MAC-T cells by resveratrol, because knockdown of Nrf2 abolished resveratrol-induced cytoprotective effects against oxidative stress, accompanied by no significant differences in gene expression of Nrf2, HO-1, Txnrd, and CHOP between resveratrol+H₂O₂ and H₂O₂ groups in Nrf2 knockdown MAC-T cells. Finally, by using selective inhibitors, we confirmed that the induction of Nrf2 by resveratrol was mediated through the activation of the PI3K/Akt and ERK/MAPK pathways, but negatively regulated by p38/MAPK pathway. In conclusion, our study provided evidence that resveratrol may be potentially used as a therapeutic agent for cytoprotection of bMEC against oxidative stress.