Glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) is a member of the tumor necrosis factor receptor (TNFR) family that is expressed at low levels on unstimulated T cells, B cells, and macrophages. Upon activation, CD4⁺ and CD8⁺ T cells up-regulate GITR expression, whereas immunoregulatory T cells constitutively express high levels of GITR. Here, we show that GITR may regulate alloreactive responses during graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). Using a BMT model with major histocompatibility complex class I and class II disparity, we demonstrate that GITR stimulation in vitro and in vivo enhances alloreactive CD8⁺CD25⁻ T cell proliferation, whereas it decreases alloreactive CD4⁺CD25⁻ proliferation. Allo-stimulated CD4⁺CD25⁻ cells show increased apoptosis upon GITR stimulation that is dependent on the Fas–FasL pathway. Recipients of an allograft containing CD8⁺CD25⁻ donor T cells had increased GVHD morbidity and mortality in the presence of GITR-activating antibody (Ab). Conversely, recipients of an allograft with CD4⁺CD25⁻ T cells showed a significant decrease in GVHD when treated with a GITR-activating Ab. Our findings indicate that GITR has opposite effects on the regulation of alloreactive CD4⁺ and CD8⁺ T cells.