A high plasma concentration of non-esterified fatty acids (NEFAs) is an important pathogenic factor that leads to ketosis and fatty liver in dairy cows. NEFAs may be associated with oxidative stress in dairy cows with ketosis or fatty liver and the subsequent induction of hepatocyte damage. However, the molecular mechanism of NEFAs-induced oxidative stress and whether NEFAs cause apoptosis of hepatocytes are unclear. Therefore, the aim of this study was to investigate the molecular mechanism of NEFAs-induced oxidative liver damage in bovine hepatocytes. The results showed that NEFAs increased oxidative stress, resulting in p38 phosphorylation. High activated p38 increased the expression, nuclear localization and transcriptional activity of p53 and decreased the nuclear localization and transcriptional activity of Nrf2 in bovine hepatocytes treated with high concentrations of NEFAs. High concentrations of NEFAs also promoted the apoptosis of bovine hepatocytes. Both N-acetyl-L-cysteine (NAC) and glucose (GLU) could attenuate the NEFA-induced apoptotic damage. These results indicate that NEFAs activate the ROS-p38-p53/Nrf2 signaling pathway to induce apoptotic damage in bovine hepatocytes.