Objective: To elaborate the specific mechanism of chestnut polysaccharides beneficial to spermatogenesis. Design: 16s rRNA-based study Setting and population: Busulfan and chestnut polysaccharides treatment mouse Methods: Intestinal microbiota of different groups (n = 10) were characterized by 16S rRNA gene sequencing of the V3-V4 region. Hematoxylin eosin staining (H&E), Immunochemistry (IHC) and Real-time fluorescence quantitative PCR (RT-qPCR) were acted as methods to detect the gene expression changes in tissue. Main outcome measure: spermatogenesis Results: Compared with the Vehicle group, germ cell quantity, intestinal structure and tight junction of intestinal tissue were altered in the Busulfan-treated mice, and the intestinal microbiota was also disturbed at several levels, including phylum and genus. Busulfan disturbed the homeostasis of the intestinal microenvironment. Chestnut polysaccharides could rescue this aberrance and improve spermatogenesis via steroid hormone synthesis process. Conclusions: CPs can change gut structure and tight junctions to rescue Busulfan-impaired spermatogenesis by altering the composition of different groups of gut microbes, a rescue process that is most likely related to hormone synthesis processes. Funding: This work was supported by the Shenzhen Sanming Project, Agricultural Breeding Engineering and Major Agricultural Application Technology Innovation Project and Taishan Scholar Construction Foundation of Shandong Province of China. Key words: Chestnut polysaccharides, Spermatogenesis, Gut microbiota,16S rRNA