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Polysaccharides from the species of Boletaceae (Boletales, Agaricomycetes, Basidiomycota) are economically significant to both functional foods and medicinal industries. The crude polysaccharide from Butyriboletus pseudospeciosus (BPP) was prepared, and its physicochemical properties were characterized through the use of consecutive experimental apparatus, and its impact on the gut microbiota of Kunming mice was evaluated. Analyses of the structure characteristics revealed that BPP was mainly composed of Man, Glc, and Gal, possessing the pyranose ring and β/α-glycosidic linkages. TG analysis exhibited that BPP had great heat stability. The SEM observation performed demonstrated that BPP appeared with a rough, dense, and porous shape. Through the BPP intervention, the serum and fecal biochemical index in mice can be improved obviously (p < 0.05). The abundance of beneficial microbiota in the BPP-treated group was significantly increased, while the abundance of harmful microbiota was significantly decreased (p < 0.05). Based on the Tax4Fun, we also revealed the relationship between the species of gut microbiota and showed that the high dose of BPP has significantly changed the functional diversities compared with those in other groups (p < 0.05). The results suggest that B. pseudospeciosus could serve as potential functional food or medicine.