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Epidermal growth factor (EGF) plays a role in male germ cell development, but the precise function is yet to be defined.The present study shows that EGF stimulates rat spermatogonial proliferation in a dose-dependent manner and significantly increased the protein levels ofphosphated c-Src (p-c-Src) and phosphated signal transducer and activator of transcription 3 (p-STAT3).Moreover, over-expression of c-Src tagged with enhanced green fluorescence protein (EGFP) in rat spermatogonial stem cells enhances the cell viability.In contrast, knockdown or inhibition of c-Src inhibits rat spermatogonial stem cell proliferation; EGF could not abrogate the inhibitory effect.Evidently, the content of p-STAT3 protein was increased in c-Src expressing cells and decreased in c-Src suppressing cells.Furthermore, knockdown or inhibition of STAT3 also suppressed cell viability; neither EGF nor increased c-Src could reverse the inhibitory effect.These results are the first evidence that EGF induces proliferation of rat spermatogonial stem cells via c-Src/STAT3 signal.