Self-renewal and differentiation of spermatogonial stem cell (SSC) are critical for male fertility and reproduction, both of which are highly regulated by testicular microenvironment. Exosomal miRNAs have emerged as new components in intercellular communication. However, their roles in the differentiation of SSC remain unclear. Here, we observed miR-486-5p enriched in Sertoli cell and Sertoli cell-derived exosomes. The exosomes mediate the transfer of miR-486-5p from Sertoli cells to SSCs. Exosomes release miR-486-5p, thus up-regulate expression of Stra8 (stimulated by retinoic acid 8) and promote differentiation of SSC. And PTEN was identified as a target of miR-486-5p. Overexpression of miR-486-5p in SSCs down-regulates PTEN expression, which up-regulates the expression of STRA8 and SYCP3, promotes SSCs differentiation. In addition, blocking the exosome-mediated transfer of miR-486-5p inhibits differentiation of SSC. Our findings demonstrate that miR-486-5p acts as a communication molecule between Sertoli cells and SSCs in modulating differentiation of SSCs. This provides a new insight on molecular mechanisms that regulates SSC differentiation and a basis for the diagnosis, treatment, and prevention of male infertility.