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The effects of FSH on the proliferation of sertoli cells of new b calves were studied in order to provide some data for theoretical research and practical use of spermatogenesis in vitro. Different concentrations of FSH (0, 0.01, 0.02, 0.04, and 0.08 IU? mL-1) were taken to treat bovine sertoli cellsin vitro culture, the number of sertoli cells and the expression of seven genes were determined at 6, 12 and 24 h after FSH treatments. FSH could significantly promote the proliferation ofin vitrocultured sertoli cells. FSH had no significant effects on the expression of CDC25A and could significantly improve the expression of CDC25B. 0.04 IU? mL-1 and 0.08 IU? mL-1 FSH treatments decreased the expression of CDC25C at 12 h. 0.08 IU? mL-1 FSH treatment decreased the expression of CDC25C at 24 h. 0.04 IU? mL-1 FSH could significantly decrease the expression of GSK-3β and improve the expression ofβ-catenin at 6, 12 and 24 h. 0.02, 0.04 and 0.08 IU? mL-1 FSH treatments enhanced the expressions of CYCLIND1 and C-MYC. In conclusion, FSH promoted the proliferation of sertoli cells and 0.04 IU? mL-1 FSH concentration could significantly promote the proliferation ofin vitrocultured sertoli cells. FSH promoted the proliferation of sertoli cells by CDC25B and WNT/β-catenin and CDC25B might be the key regulator to the proliferating rate of sertoli cells of bovine calf.