Abstract Azacitidine (AzaC) mitigates graft-versus-host disease (GvHD) in both murine preclinical transplant models and in human clinical trials while maintaining a robust graft-versus-leukemia effect. Previous studies have failed to investigate the role of natural regulatory T cells (nTregs) on the mitigation of GvHD by AzaC, instead focusing on the generation of suppressive Tregs (CD4+CD25+FOXP3+) through the in vivo conversion of alloreactive donor T effectors (Teffs; CD4+CD25−FOXP3−) and the direct antiproliferative effects of AzaC on allogeneic T cells. Using B6.Foxp3DTR/GFP mice in which Tregs can be specifically ablated through administration of diphtheria toxin, we demonstrate that natural Tregs are required in the donor graft for AzaC to optimally protect against GvHD and that nTregs, unlike Teffs (CD3+FOXP3−), are resistant to the antiproliferative effects of AzaC. Gene expression analysis identified the potent cell cycle inhibitor, p21, was significantly upregulated in Teffs but not nTregs after treatment with AzaC. Furthermore, we demonstrate that Teffs deficient in p21 are less sensitive to the antiproliferative effects of AzaC. These results demonstrate that nTregs are essential for AzaC to fully protect against GvHD and have important clinical implications for future clinical trials testing AzaC as a novel method of GvHD prophylaxis in man.