The vacuolar-type H+-ATPase (v-ATPase) is a proton pump acidifying intracellular compartments, among them endosomes, lysosomes or clathrin-coated vesicles, in order to regulate various cellular functions. The v-ATPase has increasingly gained attention as a promising therapeutic target for the treatment of diseases such as osteoporosis or cancer during the last decades. In this study we focus on the in vitro effects of the natural product archazolid, a specific inhibitor of the v-ATPase first isolated from the myxobacterium Archangium gephyra, in the vascular endothelium. The endothelium represents an important barrier regulating cell-cell interactions. In particular with regard to pathophysiological conditions, we focused on endothelial/cancer cell interactions in vitro. We found that archazolid concentrations up to 1 nM did not impair the viability of human umbilical vein endothelial cells (HUVECs) but successfully inhibit the v-ATPase resulting in an abrogated lysosomal acidification. In in vitro experiments the adhesion of the breast cancer cell line MDA-MB-231 to an archazolid-treated HUVEC monolayer was significantly increased. Of note, the involvement of prominent cell adhesion molecules playing a crucial role in cancer cell adhesion onto the endothelium could be excluded. Interestingly, blocking of beta1 integrins on MDA-MB-231 cells abrogated the archazolid-induced cancer cell adhesion onto HUVECs. In line with these results, we found that archazolid increased the expression of collagen on the endothelial surface, the favored ligand of beta1 integrin on MDA-MB-231 cells. This effect was accompanied by a significantly reduced expression and activity of the lysosomal proteinase cathepsin B. Over-expression of cathepsin B reversed the pro-adhesive effect in endothelial cells. This study shows for the first time that the archazolid-induced v-ATPase inhibition in endothelial cells modifies the interactions between endothelial and cancer cells by a pro-adhesive phenotype. Further approaches considering v-ATPase inhibitors as therapeutic strategy for the treatment of pathophysiological conditions should take our findings into account.