Polysaccharides with low toxicity and high biological activities are a kind of biological macromolecule. Recently, growing studies have confirmed that polysaccharides could improve obesity, diabetes, tumors, inflammatory bowel disease, hyperlipidemia, diarrhea, and liver-related diseases by changing the intestinal micro-environment. Moreover, polysaccharides could promote human health by regulating gut microbiota, enhancing production of short-chain fatty acids (SCFAs), improving intestinal mucosal barrier, regulating lipid metabolism, and activating specific signaling pathways. Notably, the biological activities of polysaccharides are closely related to their molecular weight, monosaccharide composition, glycosidic bond types, and regulation of gut microbiota. The intestinal microbiota can secrete glycoside hydrolases, lyases, and esterases to break down polysaccharides chains and generate monosaccharides, thereby promoting their absorption and utilization. The degradation of polysaccharides can produce SCFAs, further regulating the proportion of gut microbiota and achieving the effect of preventing and treating various diseases. This review aims to summarize the latest studies: 1) effect of polysaccharides structures on intestinal flora; 2) regulatory effect of polysaccharides on gut microbiota; 3) effects of polysaccharides on gut microbe-mediated diseases; 4) regulation of gut microbiota on polysaccharides metabolism. The findings are expected to provide important information for the development of polysaccharides and the treatment of diseases.