The present study was conducted to observe the protective effects of c9, t11- conjugated linoleic acid (CLA) on oxidative stress and inflammation in bovine mammary epithelial cells (BMECs) exposed to H2O2. The BMECs were treated with different concentrations of H2O2 for 8 h, 600 µmol/L was determined to be the damage concentration. Using different concentrations of c9, t11-CLA to process BMECs for 24 h, 50 and 100 µmol/L were determined to be the effective concentrations for subsequent analyses. Thus, four BMEC groups were established: Control group; H2O2 group; 50 µmol/L c9, t11-CLA + H2O2 group; 100 µmol/L c9, t11-CLA + H2O2 group. We observed that the H2O2 group exhibited significantly lower total antioxidant activity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities and significantly higher secretions of malondialdehyde (MDA), interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α and expressions of IL-1β, IL-6, and IL-8 than the control group (p < 0.05). Pretreatment with c9, t11-CLA enhanced SOD, CAT, and GPx activities and SOD mRNA expression and repressed IL-6 and IL-8 secretion and expression in H2O2-treated BMECs (p < 0.05). In conclusion, c9, t11-CLA treatment efficiently enhanced antioxidant capacity and decreased inflammation induced by H2O2 in BMECs.