Culture of mouse spermatogonial stem cells (mSSCs) contributes to understanding the mechanisms of mammalian spermatogenesis. Several key growth factors such as GDNF and FGF2 have been known to be essential for the proliferation of cultured mSSCs. However, additional factors regulating SSC proliferation remain to be identified. In this study, we report that IGF-1R signaling is required for the proliferation of cultured mSSCs by promoting the G2/M progression of the cell cycle. IGF-1 and its receptor IGF-1R are expressed in cultured mSSCs as well as in isolated Sertoli cells and interstitial cells. Blockage of IGF-1R signaling either by knockdown of IGF-1R or by the IGF-1R-specific inhibitor picropodophyllin (PPP) significantly reduced the proliferation of mSSCs, increased their apoptosis, and impaired their stem cell activity in an insulin-independent manner. PPP treatment of mSSCs blocked the G2/M progression. In contrast, both GDNF withdrawal and FGF2 signaling blockade decreased the entry of mSSCs into their S phases. Consistently, IGF-1 promoted the G2/M progression of thymidine-treated mSSCs, which were arrested at G1/S boundary synchronously; while GDNF and/or FGF2 stimulated their entry into the S phase. Moreover, IGF-1 activated the phosphorylation of AKT but not that of ERK1/2 in mSSCs. These results indicate that IGF-1R signaling stimulates the proliferation of mSSCs using a distinct mechanism from those by GDNF and FGF2, and will contribute to the establishment of a chemically defined culture system.