OBJECTIVE We found seminal B7-H3 was associated with human sperm concentration. However, the mechanism is unclear. The purpose of this study was to investigate the expression of B7-H3 in mouse testis and determine the effects of B7-H3 on the proliferation of mouse spermatogonial stem cells (SSCs) and the underlying mechanisms. METHODS B7-H3 expression in the testis of mice at different ages (3 weeks, 8 weeks, 4 months and 9 months) was detected by western blot and immunohistochemistry. CCK-8 were used to measure mouse SSCs proliferation after incubation with different concentrations of B7-H3 for 1-72 h in vitro. Flow cytometry was used to analyze the cell cycle of mouse SSCs after incubation with different concentrations of B7-H3 for 48 and 72 h. The signaling pathways involved were assessed by western blot. RESULTS Four-month-old mice had the highest expression of B7-H3 in the testis, while 3-week-old mice had the lowest expression of B7-H3. B7-H3 was predominantly detected on the membrane and in the cytoplasm of Sertoli cells. Furthermore, B7-H3 promoted mouse SSCs proliferation and increased the percentage of cells in S+G2/M phase in a time- and dose-dependent manner in vitro. These effects were inhibited by LY294002, indicating the involvement of the phosphoinositide 3-kinase signaling pathway. CONCLUSIONS The expression of B7-H3 in mouse testis, especially Sertoli cells, was associated with mouse age. In vitro, B7-H3 promoted the proliferation and accelerated the cell cycle of mouse SSCs via the PI3K pathway, indicating a critical role of B7-H3 expressed by Sertoli cells in mouse spermatogenesis.