The objective of this study was to investigate hepatic oxidative stress and the apoptotic status of ketotic cows, and the mechanism of acetoacetate (AcAc)-induced hepatic oxidative damage in ketotic cows. Liver and blood samples were collected from healthy and clinically ketotic cows. Hepatocytes were isolated from calves and treated with 0.3, 1.2, 2.4 or 4.8 mM AcAc in presence or absence of antioxidant N-acetylcysteine (NAC), JNK inhibitor SP600125, p38MAPK inhibitor SB203580, and ERK activator SC3527, respectively. Compared with healthy cows, ketotic cows exhibited severe hepatic injury and oxidative stress. The blood concentration of the apoptotic marker CK18 M30 and the number of TUNEL-positive cells in the liver of ketotic cows were 1.19- and 2.61-fold, respectively, higher than the values observed in control cows. Importantly, the levels of phosphorylated c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK) were significantly increased but the level of phosphorylated extracellular signal-regulated kinase1/2 (ERK1/2) was markedly decreased, which further promoted tumor protein 53 (p53) expression and inhibited nuclear factor E2-related factor 2 (Nrf2) expression. In vitro, AA treatment increased reactive oxygen species (ROS) content and further induced oxidative stress and apoptosis of calf hepatocytes. AA treatment increased the phosphorylation levels of JNK and p38MAPK and decreased the phosphorylation level of ERK, which could increase p53 and inhibit 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 NAC treatment or interference of MAPKs pathway could attenuate the hepatocytes apoptosis induced by AA. Collectively, ketotic cows display severe hepatic oxidative stress and the hepatic MAPK-p53/Nrf2 apoptotic pathway is over induced and partially mediated apoptotic damage. Besides, AA triggers hepatocytes apoptosis via the ROS-mediated MAPKs pathway in ketotic cows.