Tumor development involves complex bidirectional interactions between tumor cells and host stromal cells. Endosialin (Tem1) has been identified as a highly O-glycosylated transmembrane glycoprotein, which is specifically expressed by tumor vessel-associated pericytes and stromal fibroblasts of a wide range of human tumors. Recent experiments in endosialin-deficient mice have unraveled a critical role of endosialin in site-specific tumor progression and metastasis. To molecularly understand the mechanisms of endosialin function, we aimed to identify extracellular endosialin ligands and identified Mac-2 BP/90K as a specific interaction partner. Detailed biochemical analyses identified a C-terminal fragment of Mac-2 BP/90K, which was shown to contain binding sites for galectin-3, and collagens as the structures responsible for endosialin binding. Subsequent expression analysis of Mac-2 BP/90K in vivo revealed weak or no expression in most normal tissues and strong up-regulation in tumor cells of human neoplastic tissues. Intriguingly, the expression patterns of Mac-2 BP/90K and endosialin were mutually exclusive in all human tissues. Correspondingly, loss-of-function adhesion experiments of Mac-2 BP/90K-expressing tumor cells on endosialin-expressing fibroblasts revealed a repulsive outcome of the Mac-2 BP/90K interaction. Taken together, the experiments identify a novel repulsive interaction between endosialin on stromal fibroblasts and Mac-2 BP/90K on tumor cells.—Becker, R., Lenter, M. C., Vollkommer, T., Boos, A. M., Pfaff, D., Augustin, H. G., Christian, S. Tumor stroma marker endosialin (Tem1) is a binding partner of metastasis-related protein Mac-2 BP/90K.