T cell stimulation via glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) can evoke effective tumor immunity. A single administration of agonistic anti-GITR monoclonal antibody (mAb) to tumor-bearing mice intravenously or directly into tumors provoked potent tumor-specific immunity and eradicated established tumors without eliciting overt autoimmune disease. A large number of CD4+ and CD8+ T cells, including interferon (IFN)-gamma-secreting cells, infiltrated regressing tumors. Tumor-specific IFN-gamma-secreting CD4+ and CD8+ T cells also increased in the spleen. The treatment led to tumor rejection in IFN-gamma-intact mice but not IFN-gamma-deficient mice. Furthermore, coadministration of anti-GITR and anti-CTLA-4 mAbs had a synergistic effect, leading to eradication of more advanced tumors. In contrast, coadministration of anti-CD25 and anti-GITR mAbs was less effective than anti-GITR treatment alone, because anti-CD25 depleted both CD25+-activated effector T cells and CD25+CD4+ naturally occurring regulatory T (T reg) cells. Importantly, CD4+ T cells expressing the T reg-specific transcription factor Foxp3 predominantly infiltrated growing tumors in control mice, indicating that tumor-infiltrating natural Foxp3+CD25+CD4+ T reg cells may hamper the development of effective tumor immunity. Taken together, T cell stimulation through GITR attenuates T reg-mediated suppression or enhances tumor-killing by CD4+ and CD8+ effector T cells, including those secreting IFN-gamma, or both. Agonistic anti-GITR mAb is therefore instrumental in treating advanced cancers.