Radix Puerariae thomsonii, the root of the botanical family Fabaceae species Pueraria montana var. thomsonii (Benth.) MR Almeida, can be used as food or medicine. Polysaccharides are important active constituents of this root. A low molecular weight polysaccharide, RPP-2 having α-D-1,3-glucan as the main chain, was isolated and purified. RPP-2 could promote the growth of probiotics in-vitro. Therefore, the effects of RPP-2 on a high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) C57/BL6J mouse models were investigated. RPP-2 could reduce HFD-induced liver injury by reducing inflammation, glucose metabolism, and steatosis, thereby improving NAFLD. RPP-2 regulated the abundances of intestinal floral genera Flintibacter, Butyricicoccus, and Oscillibacter, and their metabolites Lipopolysaccharide (LPS), bile acids, and short-chain fatty acids (SCFAs), thereby improving inflammation, lipid metabolism, and energy metabolism signaling pathways. These results confirmed that RPP-2 play a prebiotic role by regulating intestinal flora and microbial metabolites, playing a multi-pathway and multi-target role in improving NAFLD.