To explore the protective effect of gentiopicroside (GPS) on non-alcoholic fatty liver (NAFLD) rats and the effects of TLR-4/NF-κB and AMPK/Nrf2 pathways.Sixty SD rats were randomly divided into normal group,model group,metformin group (200 mg/kg) and GPS high,medium and low dose groups (120,60,30 mg/kg).The normal group was fed standard diet and the other groups were fed high-fat diet for 14 weeks to establish NAFLD model.Biochemical methods were used to detect the levels of liver function,oxidative stress and lipid accumulation.Enzyme-linked immunosorbent assay (ELISA) was used to detect insulin resistance and inflammatory factor levels.Western blot was used to detect the expression of TLR-4/NF-κB and AMPK/Nrf2 pathway proteins.Oil red O staining was used to observe the pathological changes of liver tissue.The results shows GPS reduces the activity or content of alanine aminotransferase (ALT),aspartate aminotransferase (AST),malondialdehyde (MDA),cholesterol (TC),triglyceride (TG),total low density lipoprotein cholesterol (LDL-C),fasting blood glucose (FBG),fasting insulin (FINS) and insulin resistance index (HOMA-IR),enhances the activity of superoxide dismutase (SOD),glutathione peroxidase (GSH-Px) and high density lipoprotein cholesterol (HDL-C),reduces the levels of interleukin-1β(IL-1β),interleukin-6 (IL-6),Tumor necrosis factor-α(TNF-α),NF-κBp-p65 and TLR-4 in the liver,increase the expression levels of p-AMPK and Nrf2.In conclusion,the mechanism of GPS to improve NAFLD may be related to inhibition of oxidative stress,inflammatory and regulation of TLR-4/NF-κB and AMPK/Nrf2 signaling pathways.