Hepatocyte cell death is a characteristic indication in the development of non-alcoholic steatohepatitis (NASH); however, the underlying mechanism is still unclear. In this study, we examined the potential mechanism(s) involved in the development of liver injury using a methionine-choline deficient (MCD) diet feeding NASH model. Male C57BL6/J mice were fed MCD and methionine-choline sufficient (MCS) diet for two weeks before being killed. Our results showed that MCD diet feeding resulted in fatty liver and liver injury, evidenced by increased hepatic triglyceride (TG), plasma alanine aminotransferases and hepatic thiobarbituric acid reactive substances levels in MCD-fed mice. Furthermore, we found that MCD diet feeding caused remarkable suppression of hepatic extracellular signal-regulated kinases (ERK) 1/2 activation and increased transforming growth factor (TGF)-beta1 levels in plasma and the liver tissue. In vitro investigations showed that intracellular MEK/ERK1/2 activation status played a critical role in the determination of sensitivity of hepatocytes to TGF-beta1-induced cell death. HepG2 cells, otherwise resistant to TGF-beta1 killing due to high level of ERK1/2 activation, was sensitized by U0126, a specific MEK/ERK1/2 inhibitor, to TGF-beta1 cytotoxicity. H4IIEC3 cells, which have lower level of constitutive ERK1/2 activity, are sensitive to TGF-beta1-induced cell death. Lastly, we demonstrated that administration of epidermal growth factor, a strong ERK1/2 activator, to MCD-fed mice attenuated liver injury without affecting hepatic TG accumulation. Our findings demonstrated that hepatic ERK1/2 inactivation aggravates TGF-beta1-induced hepatotoxicity, which may contribute, at least in part, to the initiation of liver injury in NASH.