Estrogen, through its receptors, plays an important role in regulation of spermatogenesis. The importance of estrogen is highlighted with the reports that environmental estrogens have deleterious effects on spermatogenesis and have been associated with declining sperm counts in men. Although estrogen receptors (ERα and ERβ) have been localised in the seminiferous epithelium; their precise roles are yet unclear as in vivo estradiol treatment would signal through both the ERs. It is important to understand the effects of activation of estrogen signaling through both the receptors since several environmental estrogens have different binding affinities to the two ERs. Hence we had developed in vivo selective ER agonist administration models in adult male rats to decipher the individual roles of the ERs. Treatment with both ERα and ERβ agonists decreased sperm counts after 60 days of treatment. This study was aimed at understanding the factors contributed, by the two ERs, to the decreased sperm counts. Treatment with ERα agonist causes an arrest in differentiation of round spermatids into elongated spermatids, mainly due to down-regulation of genes involved in spermiogenesis by activation of estrogen signaling through ERα. ERβ agonist administration reduces sperm counts due to spermiation failure and spermatocyte apoptosis. Spermiation failure observed was due to defects in tubulobulbar complex formation because of decrease in expression of genes involved in actin remodelling. The increase in spermatocyte apoptosis could be due to increase in oxidative stress conditions and decrease in anti-apoptotic genes. Our results suggest that the ERs regulate distinct aspects of spermatogenesis. ERα is mainly involved with regulation of spermiogenesis, while ERβ regulates spermatocyte apoptosis and spermiation. Activation of estrogen signaling through either of the receptors can affect their respective processes during spermatogenesis and lead to low sperm output. These observations can be useful in understanding potential effect of environmental estrogens on spermatogenesis.