Abstract: The aim of this study was to evaluate the effect of alfalfa flavonoids on in vitro bovine mammary epithelial cells under heat stress. Bovine mammary epithelial cells were cultured on media with different concentrations of alfalfa flavonoids (0, 25, 50, 75, and 100 μg/mL) at 37℃ with 5% CO₂ for 72 h. Then, the cultured cells were subjected to a heat-shock treatment (42℃ for 1 h) before being returned to 37℃ with 5% CO₂ for 12 h. The cell activity, antioxidant index, and gene transcript levels were evaluated. The results showed that the cell activity was significantly higher in the 25 μg/mL treatment group than in the 0 and 50 μg/mL treatment groups (P<0.05), but cell activity did not differ significantly among the other groups. Compared with the control group (0 μg/mL) the 50, 75, and 100 μg/mL treatment groups showed significantly higher glutathione peroxidase activity (P<0.01) and significantly lower lactate dehydrogenase activity and malondialdehyde contents (P<0.01 and P<0.05, respectively). The catalase activity did not differ significantly among the five groups. Compared with the control group (0 μg/mL), the 50 and 75 μg/mL treatment groups showed reduced transcript levels of Caspase3 and Socs3, and the 25 μg/mL group showed significantly increased transcript levels of P53, Stat1, and Socs1 (P<0.01 or P<0.05). The transcript levels of Bcl-2 and Fas did not differ significantly among the groups. Together, these results show that alfalfa flavonoids can increase cell activity and antioxidant capacity and inhibit cell apoptosis, and that the optimal concentration of alfalfa flavonoids is 75 μg/mL.