BackgroundTrichomonas vaginalisis a widespread and important sexually transmitted pathogen. Adherence to the surface of the host cell is the precondition for the parasitism and pathogenicity of this parasite.Trichomonas vaginalisadhesion protein 33 (TvAP33) plays a key role in the process of adhesion, but how this protein mediates the adhesion and pathogenicity ofT. vaginalisto host cells is unclear.MethodsThe expression of TvAP33 in trophozoites was knocked down by small interfering RNA. VK2/E6E7 cells and mice infected withT. vaginaliswere used to evaluate the pathogenicity ofT. vaginalis. We constructed a complementary DNA library of VK2/E6E7 cells and screened the protein molecules interacting with TvAP33 by the yeast two-hybrid system. The interaction between TvAP33 and BNIP3 (Bcl-2 interacting protein 3) was analyzed by co-immunoprecipitation and colocalization.ResultsFollowing knockdown of TvAP33 expression, the number ofT. vaginalistrophozoites adhering to VK2/E6E7 cells decreased significantly, and the inhibition of VK2/E6E7 cell proliferation and VK2/E6E7 cell apoptosis and death induced byT. vaginaliswere reduced. Animal challenge experiments showed that the pathogenicity of trophozoites decreased following passive immunization with TvAP33 antiserum or blocking of the TvAP33 protein. Immunofluorescence analysis revealed that TvAP33 could bind to VK2/E6E7 cells. Eighteen protein molecules interacting with TvAP33 were identified by the yeast two-hybrid system. The interaction between TvAP33 and BNIP3 was further confirmed by co-immunoprecipitation and colocalization. When the expression of both TvAP33 and BNIP3 in trophozoites was knocked down by small RNA interference, the number ofT. vaginalisadhering to VK2/E6E7 cells and the inhibition of VK2/E6E7 cell proliferation were significantly lower compared to trophozoites with only knockdown of TvAP33 or only BNIP3. Therefore, the interaction of TvAP33 and BNIP3 in the pathogenesis ofT. vaginalisinfecting host cells is not unique and involves other molecules.ConclusionsOur study showed that the interaction between TvAP33 and BNIP3 mediated the adhesion and pathogenicity ofT. vaginalisto host cells, providing a basis for searching for drug targets forT. vaginalisas well as new ideas for the prevention and treatment of trichomoniasis.Graphical AbstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s13071-023-05798-x.