Previous studies have indicated that targeted depletion of Bmp8 in mice can cause the reduction of germ cells in the postnatal testis and this further leads to male infertility, indicative of its importance in the maintenance of spermatogenesis. However, except the genetic impacts, no direct signaling pathway of BMP8 in cells has been interpreted in vitro due to the lack of functional BMP8 proteins. To solve this, we firstly generated different constructs for producing the recombinant BMP8 proteins in the prokaryotic and eukaryotic expression systems. With the bioactive recombinant BMP8 proteins generated, we found that BMP8 is able to activate the BMP signaling by promoting the phosphorylation of Smad1/5/8. In the reporter assays using HEK293T cells, overexpression in combination with knockdown experiments indicated that ALK3 and ALK6 of the type I receptors and ACVR2A and BMPR2 of the type II receptors can be the candidates to form the receptor complex for conducting BMP8-driven signaling. BMP8 was shown to be expressed in the testis of neonatal mice by immunoblotting and its expression is further localized in spermatogonia by immunofluorescence staining. In addition to characterization of the receptor identities of BMP8 in spermatogonia, we also found that BMP8 is able to induce differentiation of spermatogonia by increasing Kit expression. It also suppressed the expression of the GDNF-mediated self-renewal genes. Thus, our findings conclude that BMP8, capable of promoting spermatogonia differentiation at least, participates in the completion of spermatogenesis.