Elevated plasma nonesterified fatty acids (NEFA) and hepatocytes damage are features of ketosis in dairy cows.Oxidative stress is involved in the pathogenesis of liver damage induced by NEFA.However, the exact mechanism between reactive oxygen species (ROS) and the liver apoptosis injury induced by NEFA remain poorly understood.It is well accepted that apoptosis induced cellular damage by oxidative stress.In the present study, we identified the signaling transduction pathway in bovine hepatocytes responsible for apoptosis induced by NEFA.The results demonstrated that NEFA depleted intracellular glutathione (GSH) , increased the content of malondialdehyde (MDA), contributed to ROS generation, transcriptional activation of p53, transcriptional inhibition of Nrf2, loss of mitochondrial membrane potential (MMP), release of apoptosis-inducing factor (AIF)and cytochrome (cyt c) to cytosol, leading to hepatocytes apoptosis.In addition, NEFA triggered apoptosis in bovine hepatocytes via the regulation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated protein kinases (ERK1/2), Bcl-2-associated X protein (Bax), B-cell lymphonma gene 2 (Bcl-2), caspase 9 and poly ADP-ribose polymerase (PARP).Pre-treatment with inhibitors SP600125, PD98059, and antioxidants Nacetylcystein (NAC) indicated that NEFA-ROS-JNK/ERK-mitochondrial signaling pathway plays a crucial role in hepatocytes apoptosis induced by NEFA.Meanwhile, the results suggested that transcription factors p53 and Nrf2 is the downstream of NEFA-ROS-JNK/ERK and involved in hepatocytes apoptosis induced by NEFA.In conclusion, these findings supported that NEFA-ROS-JNK/ERK-mitochondrial pathway plays an important role in NEFA-induced bovine hepatocytes apoptosis and strongly imply a new clue that the inhibitors of SP600125,PD98059 ,and NAC maybe developed as medicine for prevention hyperlipidemia-induced apoptosis damage in ketotic dairy cows.