Abstract: The aim of this study was to examine the effect of alfalfa flavonoids (AF) on apoptosis of bovine mammary epithelial cells (BMECs) induced by lipopolysaccharide (LPS). The BMECs were exposed to 4 treatments; medium containing 0 μg·mL^-1 LPS and AF (control), 1 μg·mL^-1 LPS (L), 1 μg·mL^-1 LPS and 75 μg·mL^-1 AF(L+F), and 75 μg·mL^-1 AF(F), respectively. The BMECs were cultured in cell incubator at 37 ℃, 5% CO₂. The results were as follow: 1) AF supplementation significantly reduced the viability of BMECs stimulated by LPS for 12 h (P<0.01). 2) LPS significantly increased the concentration of ROS in cells (P<0.01), whereas AF supplementation significantly reduced the concentration of ROS in cells induced by LPS (P<0.05). 3) LPS significantly increased the relative expression of IL-1β, IL-6, TNF-α, TLR2, TLR4 and MyD88 in cells (P<0.01), but the relative expression of IL-1β, IL-6, TNF-α and TLR2 in cells induced by LPS was significantly reduce by AF supplementation (P<0.01 or P<0.05). 4) LPS significantly increased the expression of p53, Caspase3, p38 and P-p38 proteins in cells (P<0.01 or P<0.05), whereas AF supplementation inhibited the expression of p53 and p38 proteins in cells induced by LPS (P<0.05). The results showed that AF could improve the viability of cells and inhibit apoptosis by reducing the concentration of ROS and that AF might play a role in protecting cells against inflammatory injure by inhibiting the TLR2/MyD88 signaling pathway.