Spermatogenesis is a precisely controlled and timed process, comprising mitotic divisions of spermatogonia, meiotic divisions of spermatocytes, maturation and differentiation of haploid spermatids giving rise to spermatozoa. It is well known that the maintenance of spermatogenesis is controlled by gonadotrophins and testosterone, the effects of which are modulated by a complex network of locally produced factors, including oestrogens. However, it remains uncertain whether oestrogens are able to activate rapid signalling pathways directly in male germ cells. Classically, oestrogens act by binding to oestrogen receptors (ESRs) 1 and 2. Recently, it has been demonstrated that rapid oestrogen action can also be mediated by the G-protein-coupled oestrogen receptor 1 (Gper). The aim of the present study was to investigate ESRs and Gper expression in primary cultures of adult rat round spermatids (RS) and define if oestradiol (E2) is able to activate, through these receptors, pathways involved in the regulation of genes controlling rat RS apoptosis and/or maturation. In this study, we demonstrated that rat RS express ESR1, ESR2 and Gper. Short-time treatment of RS with E2, the selective Gper agonist G1 and the selective ESR1 and ERβ agonists, 4,4′,4″-(4-propyl-[1H]pyrazole-1,3,5-triyl) trisphenol (PPT) and 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), respectively, determined activation of Extra-cellular signal-regulated kinase (ERK1/2) through the involvement of epidermal growth factor receptor transactivation. In addition, we investigated the effects of ESRs and Gper pathway activation on factors involved in RS maturation. Expression of cyclin B1 mRNA was downregulated by E2, G1 and PPT, but not by DPN. A concomitant and inverse regulation of the pro-apoptotic factor Bax mRNA expression was observed in the same conditions, with DPN being the only one determining an increase in this factor expression. Collectively, these data demonstrate that E2 activates, through ESRs and Gper, pathways involved in the regulation of genes controlling rat RS apoptosis and differentiation such as cyclin B1 and Bax.