The aim of this study was to investigate the structural characteristics and in vitro fermentation patterns of polysaccharides from Boletus mushrooms. Polysaccharides were solubilized from fruit bodies of selected mushrooms Boletus auripes, B. bicolor, and B. griseus using subcritical water extraction. Boletus polysaccharides were characterized for their general physicochemical pattern, constituent monosaccharides and molecular weight. A simulated in vitro fermentation model was used to study the utilization of Boletus polysaccharides by the gut microbiota and their consequent modulation of microbial communities. Results showed that the main constituent monosaccharides of Boletus polysaccharides were glucose, galactose and mannose, followed by fucose, xylose and rhamnose, with glucose being the most abundant. The polysaccharides from B. bicolor and B. griseus exhibited a relatively high proportion of galactose and mannose, respectively. Boletus polysaccharides exhibited a wide range of molecular weights (5 kDa to 2000 kDa), which covered multiple polysaccharide populations, but the proportions of these populations varied among the samples. Boletus polysaccharides were gradually utilized by the human fecal microbiota, promoting the production of SCFAs. Boletus polysaccharides contributed to a healthier gut microbiota composition by increasing the relative abundance of beneficial bacterial genera such as Bacteroides and Faecalibacterium and reducing the relative abundance of harmful bacterial genera such as Sutterella and Escherichia-Shigella. B. bicolor polysaccharides showed better fermentability and prebiotic effects than the other Boletus polysaccharide groups. Therefore, the consumption of select Boletus mushrooms, particularly B. bicolor, could be a potential approach to obtain polysaccharides for microbiota modulation and to support gut health.