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Lactobacillis and estrogens are essential in vaginal homeostasis. In the present study, we investigated the potential direct effect of 17β-estradiol on a vaginal strain of Lactobacillus cristapus, the major bacterial species of the vaginal microbiota. 17β-estradiol (10− 6 to 10− 10 M) has no effect on the growth of L. crispatus but markedly affects its membrane dynamic. This effect appears adaptative and coherent with a signal transduction process. The surface polarity and aggregation potential of the bacteria remains unchanged after exposure to 17β-estradiol but its mean size is significantly reduced. 17β-estradiol also promotes biosurfactant production by L. crispatus and adhesion to vaginal VK2/E6E7 cells. Conversely, the biofilm formation activity of the bacterium is marginally affected. A potential 17β-estradiol binding site was identified by bioinformatics in L. crispatus as the membrane lipid rafts associated SPFH domain containing protein. Taken together, this study reveals that 17β-estradiol can exert direct effects on L. crispatus. This process might be of importance in the vaginal environment physiology by promoting adhesion of Lactobacilli to the mucosa and providing protection against pathogens.