Hepatic lipid accumulation, inflammation and gut microbiota dysbiosis are hallmarks of non-alcoholic fatty liver disease (NAFLD), which is the leading cause of chronic liver disease with no therapeutic consensus. The aim of the present study was to elucidate the mechanism of the effects of Astragalus mongholicus polysaccharides (mAPS) on lipid metabolism, inflammation and gut microbiota in a rat model of NAFLD induced by a high-fat diet (HFD). Our results showed that mAPS and Berberine supplementation reduced HFD-induced increases in body weight, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and homeostasis model assessment of insulin resistance (HOMA-IR), and these changes were accompanied by improved histological changes in the liver. Moreover, administration of mAPS and Berberine resulted in lower levels of serum triglycerides, total cholesterol and low-density lipoprotein cholesterol (LDL-c) but higher levels of high-density lipoprotein cholesterol (HDL-c) in HFD-fed rats. mAPS and Berberine treatment markedly reduced HFD-induced hepatic lipid accumulation, which was associated with increased expression of phosphorylated- adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-α (PPAR-α) but decreased expression of sterol-regulatory element binding proteins (SREBP-1). Pretreatment with mAPS or Berberine reduced HFD-induced expression of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α). In addition, mAPS downregulated the expression of colonic and hepatic Toll-like receptor 4 (TLR4) as well as phosphorylated- nuclear factor-κB (NF-κB) and nucleotide-binding domain, leucine-rich repeat-containing receptor, pyrin domain-containing-3 (NLRP3) but upregulated the expression of zonula occludens-1 (ZO-1) and occludin in HFD-fed rats. Notably, mAPS treatment reshaped the intestinal microbiome by lowering the Firmicutes to Bacteroidetes (F/B) ratio and increasing the abundance of Proteobacteria and Epsilonbacteria. mAPS supplementation had little effect on the profile of fecal short-chain fatty acids (SCFAs), but it significantly decreased the expression of colonic and hepatic G-protein coupled receptor (GPR) 41 and 43. Therefore, mAPS supplementation ameliorates hepatic inflammation and lipid accumulation in NAFLD by modulating the gut microbiota and SCFA-GPR signaling pathways. The present study provides new evidence for mAPS as a natural active substance in the treatment of NAFLD.