To investigate the antiestrogenic effect of clomiphene citrate (CC) in male rats estrogenized with estradiol-3-benzoate (EB). Prospective experimental study. Laboratory. Adult male albino rats (Holtzman strain). CC was given alone or in combination with EB. Testicular function and steroidogenic enzyme gene expression were evaluated in control versus treated groups. EB after 30 days of treatment induced a rise in TUNEL-positive germ cells adversely affecting spermatogenesis with complete absence of elongated spermatids or sperms. CC alone had only a moderate effect. In contrast, CC+EB synergistically inflicted more adverse effects as apoptotic germ cells per tubule rose further. Significant down-regulation in expression of testicular steroidogenic enzyme genes StAR, p450scc, 3β-HSD, and p450c17 was observed. In the EB-alone group, aromatase gene expression in the testis was up-regulated but reversed in brain and liver tissues. CC alone had little modulatory effect on aromatase expression. On the other hand, CC+EB countered the EB-induced rise of aromatase expression in the testis. The above findings indicate that CC in the presence of estrogen synergistically potentiates more adverse effects in testis, inhibiting expression of upstream steroidogenic enzyme genes and leading to disruption of steroidogenesis.