The epididymis and efferent ducts play major roles in sperm maturation, transport, concentration and storage by reabsorbing water, ions and proteins produced from seminiferous tubules. Gpr48-null male mice demonstrate reproductive tract defects and infertility. In the present study, we found that estrogen receptor α (ERα) was dramatically reduced in the epididymis and efferent ducts in Gpr48-null male mice. We further revealed that ERα could be upregulated by Gpr48 activation via the cAMP/PKA signaling pathway. Moreover, we identified a cAMP responsive element (Cre) motif located at −1307 to −1300 bp in the ERα promoter that is able to interact with Cre binding protein (Creb). In conclusion, Gpr48 participates in the development of the male epididymis and efferent ducts through regulation of ERα expression via the cAMP/PKA signaling pathway.