The methionine choline-deficient (MCD) diet leads to severe liver injury similar to human nonalcoholic steatohepatitis (NASH). Autophagy has emerged as a critical lysosomal pathway that maintains cell function and survival through the degradation of cellular components such as organelles and proteins. The goal of this study was to elucidate the role of autophagy in MCD-induced steatosis, fibrosis, inflammation, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress in mice. Mice were fed with MCD diet and treated with rapamycin (an autophagy enhancer) or chloroquine (an autophagy inhibitor) for 10 weeks. Liver injury was evaluated biochemically and histologically together with hepatic gene expression analysis. Autophagic flux was impaired in livers of mice fed with MCD diet, evidenced by reduced ratio of LC3-II/LC3-I and increased protein expression of p62. It was found that autophagy activation by rapamycin attenuated MCD-induced steatosis, fibrosis, inflammation, mitochondrial dysfunction, and ER stress. By contrast, MCD mice treated with chloroquine developed more liver injury. In conclusions, the autophagic pathway plays an important protective role in MCD-induced advanced NASH. Thus, pharmacological promotion of autophagy may provide a novel therapeutic strategy for treatment of NASH.