AIM: To explore the effects of mulberry on antioxidant stress injury in rats with nonalcoholic fatty liver disease (NAFLD) induced by high-fat diet. METHODS: Sixty male Sprague-Dawley rats were randomly divided into normal group (10 rats) and model making group (50 rats, high-fat feed for 8 weeks). From the 9th week, the NAFLD rats were randomly divided into model group, intervention groups (mulberry low, moderate, and high dosage, 2.5 g/kg, 5.0 g/kg, and 10.0 g/kg, respectively), and positive control group (magnesium?isoglycyrrhizinate, the original solution diluted to 1.0 g/L, 15 mL·kg-1·d-1), 10 rats per group, and were gavaged?once daily for 6 weeks. The levels of malondiaIdehyde (MDA) and adenosine triph osphate (ATP) in liver homogenate were measured. The HE staining and the oil red O staining were used to observe the distribution of lipid droplets and the pathologic changes of liver tissue. The gene expressions of nuclear factor-E2-related factor 2 (Nrf2)-antioxidant response element (ARE) [Nrf2, heme oxygenase-1 (HO-1), γ-glutamylcysteine synthetase (γ-GCS) and glutathione S-transferase P-1 (GSTP-1)] in liver were detected by real-time qPCR. RESULTS: Compared with the normal group: the levels of MDA in liver homogenate increased. The levels of ATP, the gene expressions of γ-GCS, GSTP-1, HO-1 and Nrf2 in liver were significantly decreased (P<0.01). Liver fat deposition was obvious, liver steatosis became significant. Compared with the model group: with the increase of mulberry dosage, the MDA level in liver homogenate decreased (P<0.05), the gene expressions of GSTP-1 and HO-1 increased (P<0.05), liver steatosis gradually decreased. The liver ATP level, the gene expressions of γ-GCS and Nrf2 in moderate dosage group and high dosage group were significantly increased (P<0.05). CONCLUSION: Mulberry has a good preventive and protective effect on liver steatosis of NAFLD in rats, and may play antioxidant stress effect by affecting the expression of Nrf2-ARE related genes.