Connexin (Cx) is a complex which allows direct communication between neighboring cells via exchange of signaling molecules and eventually leads to functional harmony of cells in a tissue. The initial segment (IS) is an excurrent duct of male reproductive tract and expression of numerous genes in the IS are controlled by androgens and estrogens. The effects of these steroid hormones on gene expression in the IS during postnatal development have not extensively examined. The present research investigated expressional modulation of Cx isoforms in the IS by exogenous exposure to estrogen agonist, estradiol benzoate (EB), or androgen antagonist, flutamide (Flu), at weaning age. Two different doses of EB or Flu were subcutaneously administrated in 21-day old of male rats, and expressional changes of Cx isoforms in the adult IS were analyzed by quantitative real-time PCR. Treatment of a low-dose EB (0.015 μg/kg body weight) resulted in an increased expression of Cx31 gene and a decreased expression of Cx37 gene. A high-dose EB (1.5 μg/kg body weight) treatment caused an increase of Cx31 gene expression. Increased levels of Cx30.3 and Cx40 transcripts were observed with a low-dose Flu (500 μg/kg body weight) treatment. Treatment of high-dose Flu (50 mg/kg body weight) led to expressional increases of Cx30.3, 40, and 43 genes. Our previous and present findings suggest differential responsiveness on gene expression of Cx isoforms in the IS by androgens and estrogens at different postnatal ages.