Transforming growth factor-β (TGFβ) superfamily are critical regulators of germ cell development that act as extracellular ligands of the signal transduction pathways regulating proliferation, apoptosis and other aspects of cell behaviour. As a member of the TGF-β superfamily, growth differentiation factor 9 (GDF9) plays a critical role in ovarian follicular development and the ovulation rate in females; however, its role in the testis has not been well elucidated. The aim of this study was to investigate the expression of GDF9 and its receptor genes BMPRII and ALK5 in prepuberal bovine Sertoli cells (SCs). In addition, we assessed the effects of GDF9 on immature SCs apoptosis, the cell cycle and tight junction functions. We found that GDF9 and its receptor genes BMPRII and ALK5 were expressed in immature SCs. Exogenous GDF9 significantly promoted SCs proliferation and inhibited the apoptosis of SCs by significantly upregulating Cyclin E (cell cycle) and bcl-2 (anti-apoptosis) mRNA expression and downregulating caspase-3 (pro-apoptosis) mRNA expression. Meanwhile, exogenous GDF9 significantly decreased the value of transepithelial electrical resistance by significantly downregulating claudin-11 mRNA expression and influencing the distribution of occludin. In conclusion, this study reveals that GDF9 is a key regulator of bovine SCs through the modulation of the cell cycle, apoptosis and tight junction functions.