Maintenance of normal male fertility relies on the process of spermatogenesis which is under complex endocrine control by mechanisms involving gonadotropin and steroid hormones. Although testosterone is the primary sex steroid in males, estrogen is locally produced in the testis and plays a very crucial role in male fertility. This is evident from presence of both the estrogen receptors alpha (ERa) and beta (ERb) in the testis and their absence, as in the case of knockout mice models, leads to sterility. The present study was undertaken to understand individual roles of the two ERs in spermatogenesis and their direct contribution towards the maintenance of male fertility using receptor-subtype-specific ligands. Administration of ERa and b agonists to adult male rats for 60 days results in a significant decrease in fertility, mainly due to an increase in pre-and post-implantation loss and a concomitant decrease in litter size and sperm counts. Our results indicate that ERa is mainly involved in negative feedback regulation of gonadotropin hormones, whereas both ERs are involved in regulation of prolactin and testosterone production. Histological examinations of the testis reveal that ERb could be involved in the process of spermiation since many failed spermatids were observed in stages IX-XI following ERb agonist treatment. Our results indicate that overactivation of estrogen signaling through either of its receptors can have detrimental effects on the fertility parameters and that the two ERs have both overlapping and distinct roles in maintenance of male fertility.