Background:Bovine mammary epithelial cells after calving undergo serious metabolic challenges and oxidative stress both of which could compromise autophagy. Transcription factor EB (TFEB)-mediated autophagy is an important cytoprotective mechanism against oxidative stress. However, effects of TFEB-mediated autophagy on the oxidative stress of bovine mammary epithelial cells remain unknown. Therefore, the main aim of the study was to investigate the role of TFEB-mediated autophagy in bovine mammary epithelial cells experiencing oxidative stress. Results:H2O2 challenge of the bovine mammary epithelial cell MAC-T increased protein abundance of LC3-II, increased number of autophagosomes and autolysosomes while decreased protein abundance of p62. Inhibition of autophagy via bafilomycin A1 aggravated H2O2-induced reactive oxygen species (ROS) accumulation and apoptosis in MAC-T cells. Furthermore, H2O2 treatment triggered the translocation of TFEB into the nucleus. Knockdown of TFEB by siRNA reversed the effect of H2O2 on protein abundance of LC3-Ⅱ and p62 as well as the number of autophagosomes and autolysosomes. Overexpression of TFEB activated autophagy and attenuated H2O2-induced ROS accumulation. Furthermore, TFEB overexpression attenuated H2O2-induced apoptosis by downregulating the caspase apoptotic pathway. Conclusions:Our results indicate that activation of TFEB mediated autophagy alleviates H2O2-induced oxidative damage by reducing ROS accumulation and inhibiting caspase-dependent apoptosis.