OBJECTIVE Trichomoniasis is a common sexually transmitted disease, and adhesion of the pathogen Trichomonas vaginalis to the host vaginal cells is the first step in establishing infection. For this to happen, the pathogen has to overcome a natural protective barrier composed mostly of lactobacilli. The objective of this study was to understand the role of lactobacilli in the adhesion of T vaginalis to host cells. METHODS Adhesion assays were carried out by incubating vaginal epithelial cells (VECs) with T vaginalis and lactobacilli together and compared with non-lactobacilli recipient controls. By varying incubation parameters and testing several microbial isolates, the number of pathogens that adhered to the VECs was determined by flow cytometry. RESULTS Overall, but with few exceptions, lactobacilli caused inhibition of T vaginalis adhesion to a variable degree. Lactobacillus gasseri ATCC 9857 and CBI3 (ambiguous Lactobacillus plantarum or Lactobacillus pentosus) caused the highest level of parasite adhesion inhibition and enhancement, respectively. These isolates of Lactobacillus can profoundly alter the adhesive properties of low-adherent and high-adherent strains of T vaginalis in a dose-dependent manner. Additionally, the effects of lactobacilli on T vaginalis adhesion are strictly contact-dependent, and surface lipoglycans of T vaginalis are most likely not involved in this modulation of adhesion mediated by the bacteria. CONCLUSIONS Lactobacilli can modulate adhesion of T vaginalis by significantly modifying the natural adhesive properties of various T vaginalis strains. This study highlights the importance of considering the role of the vaginal microbiota in the pathogenesis of trichomoniasis.