Objective To investigate the role of adenosine monophosphate-activated protein kinase (AMPK) and its agonist 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) in mice with chronic stress-induced non-alcoholic fatty liver disease (NAFLD). Methods BABL/c mice were randomly divided into control group, stress group, stress plus AICAR group (ST+A group) and AICAR group. The mouse models of chronic stress was established in the stress and ST+A groups, and the mice were injected with AICAR 500 mg/kg in the ST+A and AICAR groups. Before and after treating with AICAR, the levels of pro-inflammatory cytokines (tumor necrosis factor α[TNF-α] and interferon γ[IFN-γ]) in plasma of mice were detected by ELISA, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol, triglyceride and free fatty acid in plasma were determined by automatic biochemical analyzer, the hepatic steatosis was detected by hematoxylin-eosin (H-E) staining and Oil Red O staining, and the expression of AMPK protein in liver tissues was detected by Western blotting. Results Chronic stress caused liver function damage (the levels of ALT and AST were significantly increased, P<0.01) and liver steatosis in mice, the levels of pro-inflammatory cytokines (P<0.05) and free fatty acid (P<0.01) were significantly increased and the liver AMPK protein expression was significantly decreased (P<0.01). AICAR improved the liver cell steatosis, and alleviated the changes of above indicators. Conclusion Chronic stress may induce NAFLD through AMPK signaling pathway, and AMPK agonist AICAR can alleviate NAFLD caused by chronic stress.