In the seminiferous epithelium, numerous cell interactions between Sertoli cells and Sertoli-germ cells are established by specialized proteins so as to maintain the functionality of the testis. Exogenous estrogen exposure can result in alterations to these interactions and cause pathologies, including impaired spermatogenesis and tumorigenesis. In the present study, with the aim of finding markers of the action of estrogenic compounds in the mammalian testis, we focused on investigating molecules that are linked to cellular junctions. We found that the testicular vinexin (sorbin and SH3 domain-containing protein 3, encoded by the Sorbs3 gene) pattern underwent significant changes after developmental exposure to 17beta-estradiol (E₂). Vinexin is an adaptor protein that is implicated in cell adhesion and actin-cytoskeletal reorganization. We characterized, at the protein and mRNA levels, the expression patterns of vinexin isoforms during testis development and in defined cell types from the seminiferous tubule. The protein expression patterns of vinexin-interacting proteins flotillin 1 and vinculin were also analyzed. Thus, we have identified a novel association between a vinexin isoform and germ cells, which contrasts with the predominant localization of the gamma isoform in Sertoli cells. The effects of E₂ on the testes of developmentally exposed mice were evident, with total depletion of the germ-cell-associated vinexin isoform and a noticeable decrease in Sertoli-cell-related vinexin gamma.