A83-01 is a selective inhibitor of the TGF-βtype I receptor ALK, which inhibits the TGF-β-induced epithelial-to-mesenchymal transition (EMT) via the inhibition of Smad2 phosphorylation. Previous studies have showed that A83-01 promoted somatic cellular reprogramming signiifcantly. Male germline stem cells (mGSCs), as an alternative resource of pluripotent stem cells derived adult testis, have promising valuable in clinic medicine and regeneration, however, the derivation of mGSCs was complex and dififcult. What the role A83-01 plays in promoting the proliferation of mGSCs is still unknown. In this study, combined with A83-01 and knockout serum replacement (KSR) medium, we obtained a relatively feeder- and serum-free system for mGSCs culturing in vitro and the optimal concentration of A83-01 was 0.25μmol L-1. After continuous culturing, the proliferation efifciency of undifferentiated mGSCs and differentiation capacity of mGSC were examined as well. Results showed that, A83-01 dramatically increased the number of mGSCs and AP positive colonies, and the mitosis index according to the BrdU assay. A83-01 could also increase the expression of pluripotent markers including Oct4, Klf4, Nanog and c-Myc, analyzed by real-time quantative PCR. mGSCs cultured in the optimal feeder-and serum-free system combined with A83-01 could form embryoid bodies (EBs), which consisted of three embryonic layers detected by immunolfuorescence and RT-PCR. Remarkably, the results demonstrated 0.25 μmol L-1 A83-01 could promote the proliferation of mouse mGSC colonies and maintain their undifferentiated status under feeder-and serum-free systems.