EpCAM has been described as a therapeutically relevant tumor marker. We noted an interaction between EpCAM and the tight junction protein claudin-7 and here explored the nature of this interaction and its effect on EpCAM-mediated functions. The interaction between EpCAM and claudin-7 was defined in HEK293 cells transfected with rat claudin-7 and EpCAM cDNA. Deletions of the epidermal growth factor-like and the thyroglobin repeat domains of EpCAM or the cytoplasmic domain of EpCAM or claudin-7 did not prevent the EpCAM-claudin-7 association. A chimeric EpCAM molecule with an exchange of the cytoplasmic and transmembrane domains and an EpCAM molecule with point mutations in an AxxxG motif in the transmembrane region do not associate with claudin-7. HEK cells and the rat pancreatic tumor line BSp73AS, transfected with (mutated) EpCAM and claudin-7 cDNA, revealed that the association of both molecules severely alters the functional activity of EpCAM. Claudin-7-associated EpCAM is recruited into tetraspanin-enriched membrane microdomains (TEM). The TEM-located claudin-7-EpCAM complex supports proliferation accompanied by sustained extracellular signal-regulated kinase-1/2 phosphorylation, up-regulation of antiapoptotic proteins, and drug resistance, but not EpCAM-mediated cell-cell adhesion. Enhanced motility may be supported by colocalization of claudin-7 with actin bundles, which is only seen in EpCAM-claudin-7-expressing cells. The EpCAM-claudin-7 complex strongly promotes tumorigenicity, accelerates tumor growth, and supports ascites production and thymic metastasis formation. High expression of the tumor marker EpCAM is frequently associated with poor prognosis, which could well rely on the EpCAM-claudin-7 association that prohibits EpCAM-mediated cell-cell adhesion but promotes migration, proliferation, apoptosis resistance, and tumorigenicity.