Estrogen receptor α (ERα) is essential for male fertility. Its activity is responsible for maintaining epithelial cytoarchitecture in efferent ductules and the reabsorption of fluid for concentrating sperm in the head of the epididymis. These discoveries and others have helped to establish estrogen's bisexual role in reproductive importance. Reported here is the molecular mechanism to explain estrogen's role in fluid reabsorption in the male reproductive tract. It is shown that estrogen regulates expression of the Na⁺/H⁺ exchanger-3 (NHE3) and the rate of ²²Na⁺ transport, sensitive to an NHE3 inhibitor. Immunohistochemical staining for NHE3, carbonic anhydrase II (CAII), and aquaporin-I (AQP1) was decreased in ERα knockout (αERKO) efferent ductules. Targeted gene-deficient mice were compared with αERKO, and the NHE3 knockout and CAII-deficient mice showed αERKO-like fluid accumulation, but only the NHE3 knockout and αERKO mice were infertile. Northern blot analysis showed decreases in mRNA for NHE3 in αERKO and antiestrogen-treated mice. The changes in AQP1 and CAII in αERKO seemed to be secondary because of the disruption of apical cytoarchitecture. Ductal epithelial ultrastructure was abnormal only in αERKO mice. Thus, in the male, estrogen regulates one of the most important epithelial ion transporters and maintains epithelial morphological differentiation in efferent ductules of the male, independent of its regulation of Na⁺ transport. Finally, these data raise the possibility of targeting ERα in developing a contraceptive for the male.