In rodent graft-versus-host disease (GVHD) models, anti–IL-21 neutralizing mAb treatment ameliorates lethality and is associated with decreases in Th1 cytokine production and gastrointestinal tract injury. GVHD prevention was dependent on the in vivo generation of donor-inducible regulatory T cells (Tregs). To determine whether the IL-21 pathway might be targeted for GVHD prevention, skin and colon samples obtained from patients with no GVHD or grade 2 to 4 GVHD were analyzed for IL-21 protein expression. By immunohistochemistry staining, IL-21 protein-producing cells were present in all gastrointestinal tract samples and 54% of skin samples obtained from GVHD patients but not GVHD-free controls. In a human xenogeneic GVHD model, human IL-21–secreting cells were present in the colon of GVHD recipients and were associated with elevated serum IL-21 levels. A neutralizing anti–human IL-21 mAb given prophylactically significantly reduced GVHD-associated weight loss and mortality, resulting in a concomitant increase in Tregs and a decrease in T cells secreting IFN-γ or granzyme B. Based on these findings, anti–IL-21 mAb could be considered for GVHD prevention in the clinic.