An eight-week trial was conducted to investigate the effects of sanguinarine supplementation (600 μg and 1200 μg/kg) in high-fat (crude fat: 10%) diets (HF) on the intestinal physiological function of (initial weight 50.21 ± 0.68 g), based on a basic diet (5% crude fat, CON), which were named HFLS and HFHS, respectively. The results showed that the HF diet significantly impaired the intestinal immune and physical barrier function, and disrupted the balance of the intestinal microbiota in grass carp. Compared to the HF diet, sanguinarine supplementation significantly improved the levels of serum C4, C3, AKP, IgA, and IgM, and enhanced the intestinal antioxidant capacity (, , , , , and expression were significantly up-regulated). Sanguinarine significantly down-regulated the expression of and up-regulated the expression of , , , and by inhibiting MLCK signaling molecules. Additionally, sanguinarine significantly down-regulated the expression of , , and and up-regulated the expression of , , and by inhibiting NF-κB signaling molecules, thereby alleviating intestinal inflammation caused by HF diets. Furthermore, compared to the HF diet, the abundance of Fusobacterium and in the HFHS diet increased significantly, while the abundance of Firmicutes and showed the opposite trend. In conclusion, the HF diet had a negative impact on grass carp, while sanguinarine supplementation enhanced intestinal antioxidant ability, alleviated intestinal barrier damage, and ameliorated the homeostasis of the intestinal microbiota.