Estrogen plays an important role in male reproduction. Most of the actions are mediated by estrogen receptor (ER). To investigate the profile of estrogen affecting male fertility, we firstly cloned four ERs from the male Spinibarbus denticulatus, a local economically important cyprinid fish in China. Phylogenetic tree analysis ranked the four sdERs as two distinct groups of ERalpha and beta, which could be further divided into duplicated isoforms 1 and 2, respectively. High score identities were shared between each of the duplicated isoforms. All of the four sdERs distributed in central nervous system of male fish with a quite broad spectrum. However, distribution diversity became evident between sdERalpha and sdERbeta subtypes in the peripheral tissues. Both of the two isoforms of ERbeta were detected in all seven tissues examined, while expression of sdERalpha1 was mainly limited to liver, kidney, testis and intestine and sdERalpha2 was confined to liver, heart, kidney, testis and gill. During the testis recrudescing stages, serum concentration of luteinizing hormone (LH), testosterone (T) and estradiol-17beta (E(2)) were increasing. T and LH levels in the circulation were high until the later fully recrudesced phase, while serum E(2) level was low all the time. Quantitative real-time RT-PCR analysis determined the most abundance of sdERs in pituitary where the two sdERalpha isoforms positively expressed with testis development, while sdERbeta isoforms expressed with a reverse pattern. sdERalpha1 and sdERbeta1 were the primary forms in testis. sdERalpha1 gradually increased during the recrudescence process while sdERbeta1 firstly decreased during the recrudescing stage and then positively expressed in fully recrudesced stage. Little or no signal was detected in brain. The present work provided evidence of four sdERs in male reproductive system and suggested an important role of sdERalpha1 during testis recrudescence. Pituitary contained duplicates forms of sdERalpha which may play a role in the feedback effects of estrogen on LH secretion.