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Aims The number of Sertoli cells during proliferative phase determines the fate of the germ cells in male reproductive system. A well-characterized cell line may help in better understanding of Sertoli cell biology. Hence, the present study assessed estradiol signaling in a mouse immature Sertoli cell line (MSC-1) as an alternative model in place of primary culture of Sertoli cells. Main methods In this study, we used MSC-1 cell line, derived from 10-day old mice. The cell cycle parameters were assessed, and the expression and regulation of Sertoli cell-specific secretory genes ( ABP ; androgen-binding protein ) and tight junction genes ( claudin-5 , occludin , and vimentin ) in response to estradiol was studied. Key findings The results obtained suggested the presence of both estrogen receptors (ERα and ERβ) in MSC-1 cells. In vitro scratch assay and cell-cycle analysis suggested the proliferative effects of estradiol in both time- and dose-dependent manner. The gene expression profiles of ABP , claudin-5 , and occludin showed biphasic regulation at low and high doses of estradiol. Analysis of signaling pathways suggested the activation of extracellular signal-regulated kinase (ERK) pathway with significantly increased pERK/ERK ratio ( p < 0.05). The results also suggested down regulation in the expression of mir-17 family members (mir-17, mir-20b, and mir-106a) ( p < 0.05). Significance Considering the limited number of Sertoli cell lines and long-term survival inability of primary culture of Sertoli cells, MSC-1 cells could be a potential cell line for understanding the mechanisms of various cellular events in Sertoli cells. [ABSTRACT FROM AUTHOR]