Recently there has been a continuing worldwide decrease in the quality of human spermatozoa, especially in spermatozoa motility and concentration. Many factors are involved in this decline, and great efforts have been made to rescue spermatogenesis; however, there has been little progress in the improvement of sperm quality. Chestnuts are used in traditional Chinese medicine; their major active components are chestnut polysaccharides (CPs). CPs have many biological activities but their effects on spermatogenesis are unknown. The current investigation was designed to explore the impact of CPs on spermatogenesis and the underlying mechanisms. We demonstrated that CPs significantly increased sperm motility and concentration (4-fold and 12-fold, respectively), and improved seminiferous tubule development by increasing the number of germ cells after busulfan treatment. CPs dramatically rescued the expression of important genes and proteins (STRA8, DAZL, SYCP1, SYCP3, TNP1 etc.) in spermatogenesis. Furthermore, CPs increased the levels of hormone synthesis proteins such as CYP17A1 and HSD17β1. All the data suggested that CPs improved the testicular microenvironment to rescue spermatogenesis. With CPs being natural products, they may be an attractive alternative for treating infertile patients in the future. At the same time, the deep underlying mechanisms of their action need to be explored.