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. FASEB J. 22, 3059–3067 (2008)