Dairy cows with ketosis are characterized by oxidative stress, hepatic damage, and hyperketonemia. Acetoacetate (AA) is the main component of ketone bodies in ketotic cows, and is associated with the above pathological process. However, the potential mechanism was not illuminated. Therefore, the aim of this study was to investigate the mechanism of AA‐induced hepatic oxidative damage in ketotic cows. Compared with healthy cows, ketotic cows exhibited severe oxidative stress and hepatic damage. Moreover, the extent of hepatic damage and oxidative stress had a positive relationship with the AA levels. In vitro, AA treatment increased reactive oxygen species (ROS) content and further induced oxidative stress and apoptosis of bovine hepatocytes. In this process, AA treatment increased the phosphorylation levels of JNK and p38MAPK and decreased the phosphorylation level of ERK, which could increase p53 and inhibit nuclear factor E2‐related factor 2 (Nrf2) expression, nuclear localization, and DNA‐binding affinity, thereby inducing the overexpression of pro‐apoptotic molecules Bax, Caspase 3, Caspase 9, PARP and inhibition of anti‐apoptotic molecule Bcl‐2. Antioxidant N‐acetylcysteine (NAC) treatment or interference of MAPKs pathway could attenuate the hepatocytes apoptosis induced by AA. Collectively, these results indicate that AA triggers hepatocytes apoptosis via the ROS‐mediated MAPKs pathway in ketotic cows. Excess acetoacetate induced imbalance of oxidation and antioxidation, resulting in overproduction of reactive oxygen species (ROS) in the mitochondria. High concentration of ROS increased the phosphorylation levels of p38MAPK and JNK and decreased the phosphorylation levels of ERK1/2, which could activate p53 and inhibit Nrf2 expression, nuclear localization, and DNA‐binding affinity. Consequently, the expression of pro‐apoptotic molecules Bax, Casepase9, Casepase3, and PARP was significantly upregulated and anti‐apoptotic molecules Bcl‐2 were markedly downregulated, resulting in hepatocytes apoptosis in ketotic cows.