Glucocorticoid-induced TNF-related protein (GITR) has been shown to stimulate T cell-mediated antitumor immunity in mice. However, the functional relevance of GITR and its ligand (GITRL) for non-T cells has yet to be fully explored. In addition, recent evidence suggests that GITR plays different roles in mice and humans. We studied the role of GITR-GITRL interaction in human tumor immunology and report for the first time that primary gastrointestinal cancers and tumor cell lines of different histological origin express substantial levels of GITRL. Signaling through GITRL down-regulated the expression of the immunostimulatory molecules CD40 and CD54 and the adhesion molecule EpCAM, and induced production of the immunosuppressive cytokine TGF-beta by tumor cells. On NK cells, GITR is constitutively expressed and up-regulated following activation. Blocking GITR-GITRL interaction in cocultures of tumor cells and NK cells substantially increased cytotoxicity and IFN-gamma production of NK cells demonstrating that constitutive expression of GITRL by tumor cells diminishes NK cell antitumor immunity. GITRL-Ig fusion protein or cell surface-expressed GITRL did not induce apoptosis in NK cells, but diminished nuclear localized c-Rel and RelB, indicating that GITR might negatively modulate NK cell NF-kappaB activity. Taken together, our data indicate that tumor-expressed GITRL mediates immunosubversion in humans.