Vinca alkaloids vinblastine and vincristine and some of their derivatives such as vinorelbine are widely used in therapy of leukemia and several solid tumors. Their action is associated with alterations of the mitotic spindle functions that prevent the cell cycle progression and lead to mitotic block. A number of studies show that some Vinca alkaloids inhibit CaM-target interaction. The newest microtubule inhibitor, vinflunine (Javlor), currently in clinical trials, is remarkably more active than vinblastine against a number of tumors. Moreover, vinflunine is significantly less toxic than other Vinca alkaloids. The high antitumor activity of this molecule is not well understood since it binds to tubulin with an overall affinity several-fold lower than that of vinblastine or vincristine. In this study, we examined the interaction of Ca2+-CaM with vinflunine, vinblastine, and stable tubule only polypeptide (STOP) by using a combination of thermodynamic and mass spectrometric approaches. We characterized the influence of Vinca alkaloids on Ca2+-CaM-STOP complex formation. Our results revealed different binding modes to Ca2+-CaM for vinflunine and vinblastine, highlighting that adding fluorine atoms on the cleavamine moiety of the Vinca alkaloid molecule is critical for the localization of the drug on calmodulin. We demonstrate that vinflunine is a better inhibitor for STOP binding to calmodulin than vinblastine. We suggest that vinflunine action on calmodulin can have an effect on microtubule dynamics. These data may contribute to a better understanding of the superior antitumor efficiency and lower toxicity of vinflunine.