Mastitis is acute inflammation caused by microbial infections in the mammary glands. This disease is extremely harmful to lactating mothers. The preferred clinical strategy is antibiotic treatment, but this method results in resistance and side effects. Lixisenatide, a kind of glucagon-like peptide-1 (GLP-1) receptor agonist, is typically used for the treatment of type II diabetes. It is unknown whether lixisenatide possesses a beneficial role in mastitis. In the current study, we assessed the protective effects of lixisenatide against lipopolysaccharide (LPS) stimulation in MAC-T bovine mammary epithelial cells (MECs). Our findings show that lixisenatide attenuated LPSinduced oxidative stress by reducing reactive oxygen species (ROS) production and nicotinamide adenine dinucleotide phosphate (NADPH) oxidases-1 (NOX-1) expression in MAC-T MECs. Additionally, lixisenatide inhibited LPS-induced expression and secretion of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and interleukin 1β (IL-1β). We also found that lixisenatide suppressed LPS-induced expression of matrix metalloproteinase 2 (MMP-2) and metalloproteinase 9 (MMP-9), and reduced the expression of toll-like receptor 4 (TLR4) (a typical receptor of LPS), its downstream molecule myeloid differentiation factor 88 (MyD88), and the phosphorylation of TGF β-activated kinase 1 (TAK1). Notably, lixisenatide decreased the nuclear levels of nuclear factor-κB (NF-κB) and its transcriptional activity. These findings suggest that lixisenatide might become a possible therapeutic agent for the treatment of mastitis by weakening oxidative stress and the inflammatory response in MECs.