Ligation of GITR (glucocorticoid-induced TNF receptor-related gene, or TNFRSF18) by agonist antibody has recently entered into early-phase clinical trials for the treatment of advanced malignancies. Although the ability of GITR modulation to induce tumor regression is well documented in preclinical studies, the underlying mechanisms of action, particularly its effects on CD4⁺Foxp3⁺ regulatory T cells (Treg), have not been fully elucidated. We have previously shown that GITR ligation in vivo by agonist antibody DTA-1 causes more than 50% reduction of intratumor Tregs with down modulation of Foxp3 expression. Here, we show that the loss of Foxp3 is tumor dependent. Adoptively transferred Foxp3⁺ Tregs from tumor-bearing animals lose Foxp3 expression in the host when treated with DTA-1, whereas Tregs from naïve mice maintain Foxp3 expression. GITR ligation also alters the expression of various transcription factors and cytokines important for Treg function. Complete Foxp3 loss in intratumor Tregs correlates with a dramatic decrease in Helios expression and is associated with the upregulation of transcription factors, T-Bet and Eomes. Changes in Helios correspond with a reduction in interleukin (IL)-10 and an increase in IFN-γ expression in DTA-1–treated Tregs. Together, these data show that GITR agonist antibody alters Treg lineage stability inducing an inflammatory effector T-cell phenotype. The resultant loss of lineage stability causes Tregs to lose their intratumor immune-suppressive function, making the tumor susceptible to killing by tumor-specific effector CD8⁺ T cells. Cancer Immunol Res; 1(5); 320–31. ©2013 AACR.