Many studies have indicated that estrogens could have a role in the regulation of testicular function. However, it remains uncertain whether estrogens are able to directly activate signaling pathways in male germ cells. Estrogens are synthesized by the enzyme aromatase and classically act by binding to estrogen receptors (ER) and ER. Knock-out (KO) mice for both receptor isoforms exhibit a testicular phenotype which is less severe than aromatase KO mice, suggesting the existence of an estrogen-binding receptor that may compensate for the lack of ERs. Recently, studies using estrogen-sensitive tumor cell lines have demonstrated that the G-protein-coupled receptor (GPR)-30 binds and mediates estrogen action through the activation of the EGFR/ERK/fos transduction pathway. The present study investigated the ability of 17-estradiol (E2) to activate this pathway in the mouse spermatogonial cell line (GC-1). Using the GC-1 cell line as a model system, we demonstrated that GC-1 cells express GPR30 and ER but not ER. E2, the selective GPR30 agonist G1, and the selective ER agonist PPT activated the rapid ERK1/2-fos signaling cascade. This response was abrogated by the EGFR inhibitor AG1478, ERK inhibitor PD98059 and ER inhibitor ICI182780, or by silencing GPR30 expression. Moreover, E2 and G1 up-regulated cyclin D1 expression and GC-1 cell proliferation. Our results indicate for the first time that estrogens, through a cross-talk between GPR30 and ER, activate the rapid EGFR/ERK/fos pathway which in turn stimulate mouse GC-1 cell proliferation. Further studies to elucidate the involvement of rapid estrogen signaling pathways in the regulation of male fertility are warranted.