Our previous study confirmed the beneficial effects of chestnut polysaccharides (CPs) on the spermatogenesis process, but the exact mechanism is not clear. Several studies have demonstrated the importance of balanced gut microbiota in maintaining normal reproductive function. In this study, we investigated the biological functions of CPs from the perspective of gut microbiota function, expecting to find out the specific mechanism of CPs in restoring impaired spermatogenesis. Compared with the control group, the mice treated with busulfan showed a reduced number of germ cells, structural changes in the small intestine and composition alteration in the gut microbiota at several levels, including the phylum and genus. In contrast, the number of germ cells in seminiferous tubules was significantly increased, and the structure of the small intestine and the composition of the gut microbiota were altered in the busulfan-treated mice after the CPs treatment. The 16s rRNA analysis results showed that the Firmicutes was the predominant phylum in all groups followed by Proteobacteria, Bacteroidetes, Actinobacteria, Tenericutes, Cyanobacteria and unidentified bacteria. Interestingly, the subsequent functional analysis implied that the steroid hormone biosynthesis process is the major metabolic pathway in the CPs-mediated restoration process and the experimental results confirmed this speculation. In conclusion, this study confirmed that CPs can restore the impaired spermatogenesis process by adjusting the gut microbiota and intestinal structure, which will also provide technical support and a theoretical basis for the subsequent treatment of male infertility.