Regulatory T (Treg) cells can suppress a wide variety of immune responses, including antitumor and alloimmune responses. The mechanisms by which Treg cells mediate their suppressive effects depend on the context of their activation. We previously reported that granzyme B is important for Treg cell - mediated suppression of antitumor immune responses. We therefore hypothesized that granzyme B may likewise be important for suppression of graft-versus-host disease (GVHD). We found that allogeneic mismatch induces the expression of granzyme B in mixed lymphocyte reactions and in a model of graft-versus-host disease (GVHD). However, wild-type and granzyme B - deficient Treg cells were equally able to suppress effector T (Teff) cell proliferation driven by multiple stimuli, including allogeneic antigen-presenting cells. Surprisingly, adoptive transfer of granzyme B - deficient Treg cells prevented GVHD lethality, suppressed serum cytokine production in vivo, and prevented target organ damage. These data contrast strikingly with our previous study, which demonstrated that granzyme B plays a nonredundant role in Treg cell - mediated suppression of antitumor responses. Taken together, these findings suggest that targeting specific Treg cell - suppressive mechanisms, such as granzyme B, may be therapeutically beneficial for segregating GVHD and graft-versus-tumor immune responses.