Research background. Sparassis latifolia polysaccharides can regulate lipids and cholesterol in serum and liver. However, little is known about the regulation mechanism of Sparassis latifolia polysaccharides on cholesterol metabolism and especially the causal relationship with gut microbiota regulation. This study will provide a theoretical basis for the cholesterol-lowering mechanism of SLPs and the further development of functional foods. Experimental approach. In this study, we investigated the regulation mechanism of Sparassis latifolia polysaccharides on intestinal cholesterol metabolism in high-fat and high-cholesterol diet-fed rats. Briefly, enzyme colorimetric microplate method was used to determine the content of fecal bile acid. Gas chromatography-mass spectrometry technology was used to detect the content of cholesterol and fecal alcohol in feces. HE staining method was applied to observe the changes of small intestine tissue structure. The related genes expressions of jejunum and ileum were detected by real-time fluorescent quantitative polymerase chain reaction. The related protein expressions of jejunum were studied by using western blot. High-throughput sequencing technology was used to detect the intestinal flora changes of the cecal contents. gas chromatography-mass spectrometry technology was applied to detect the content of short-chain fatty acids in the cecal contents. Results and conclusions. The results showed that Sparassis latifolia polysaccharides could improve the intestinal morphological structure and physiological indices in rats with high-fat and high-cholesterol diet. Moreover, it could improve intestinal cholesterol metabolism disorder induced by high-fat and high-cholesterol diets via reducing the levels of HMGCR, NPC1L1, ACAT2, MTP, ASBT and IBABP mRNA or protein, increasing ABCG8 mRNA expression. In addition, it could also increase the relative abundance of Bacteroides, Butyricicoccus, Parabacteroides, Parasutteerella and Alloprevotella and the short-chain fatty acids content, to comprehensively regulate the intestinal cholesterol metabolism. The metabolomics analysis found that Sparassis latifolia polysaccharides could affect lipid, carbohydrate and other related metabolites. Some biomarkers associated with cholesterol metabolism correlated significantly with the abundance of specific intestinal microbiota. Novelty and scientific contribution. These findings indicated that Sparassis latifolia polysaccharides could attenuate intestinal cholesterol metabolism disorder correlating with modulating gut microbiota and improving host metabolism. It provides theoretical support for the development of Sparassis latifolia as a new resource food.