Resistant starch (RS) is a novel type of prebiotic that exerts positive effects on lipid metabolism and intestinal flora. In this study, we investigated the effects of dietary RS on lipid metabolism and the intestinal barrier in largemouth bass (Micropterus salmoides). The experimental fish were fed either a control diet (C), a high-fat diet (H), or H diets supplemented with 0.5 %, 1.5 %, and 3 % RS (HRS0.5, HRS1.5, and HRS3.0). Dietary supplementation with 1.5 % and 3.0 % RS increased the final weight and feed utilization. Moreover, the hepatic crude protein content and the expression of genes related to lipid lipolysis were significantly higher in the HRS1.5 group compared to the H group, whereas hepatic crude lipid content and the expression of genes related to lipid synthesis were considerably lower in the HRS1.5 and HRS3.0 groups than in the H group. Additionally, hepatocyte vacuolation was alleviated in the HRS1.5 and HRS3.0 groups, and the number of liver lipid droplets was significantly decreased. Dietary supplementation with 1.5 % and 3.0 % RS downregulated the expression of pro-inflammatory factors while upregulating the expression of anti-inflammatory factors. Furthermore, analysis of gut microbiota composition revealed that RS supplementation increased the population of beneficial bacteria and short-chain fatty acid (SCFA) contents, decreased the abundance of pathogenic bacteria, and enhanced the diversity and richness of the intestinal flora. Non-targeted metabolomics analysis indicated that the levels of L-arginine and betaine were significantly higher in the HRS1.5 group, while levels of L-methionine and taurocholic acid were notably elevated in the HRS3.0 group. In conclusion, dietary supplementation with 1.5-3.0 % RS improved the balance of intestinal flora, promoted the growth of beneficial bacteria, adjusted the metabolites profile, and increased the SCFA levels. These results suggest that dietary supplementation with 1.5-3.0 % RS can restore the intestinal protective barrier, reduce hepatic lipid accumulation, and regulate lipid metabolism in largemouth bass.