Abstract Graft-versus-host disease (GvHD) is a systemic disease in which donor T cells attack the allogeneic recipient. Here we investigated the role of CD73 in GvHD induced by transplantation of BALB/c bone marrow and spleen cells into lethally irradiated C57BL/6 recipients. CD73 is an enzyme that generates extracellular adenosine, a nucleoside with anti-inflammatory and immunosuppressive activity mediated by adenosine receptors. Survival curves showed more severe GvHD in CD73-/- → CD73-/- transplants than in CD73+/+ → CD73+/+ transplants and serum IFNγ and IL-6 were higher in CD73-/- recipient mice. CD4+ and CD8+ T cells from CD73-/- donors proliferated more in vivo than those from CD73+/+ donors as assessed by CFSE dye dilution and cell numbers in spleens. However, irradiation-induced activation of dendritic cells as assessed by CD86 up regulation was similar in CD73+/+ and CD73-/- mice. In vivo anti-C57BL/6 CTL activity was higher in CD73-/- transplant recipients than in CD73+/+ recipients. Additional CD73-/- → CD73+/+ and CD73+/+ → CD73-/- transplants revealed that more severe GvHD is caused primarily by a lack of CD73 in recipients. The adenosine receptor antagonist caffeine exacerbated GvHD in CD73+/+ recipients, consistent with the idea that the protective effect of CD73 depends upon its adenosine-generating activity. In conclusion, recipient CD73 plays a primary preventive role in GvHD by regulating allogeneic donor T cell activation in an adenosine-dependent manner.