Regulatory T cells (Tregs) protect against graft-versus-host disease (GVHD), a life-threatening complication of allogeneic hematopoietic stem cell transplantation. The ectoenzyme CD39 is important for increasing the immunosuppressive function of Tregs. The rs10748643 (A → G) single-nucleotide polymorphism (SNP) in intron 1 of the human ENTPD1 gene is associated with increased proportions of CD39 Tregs. This study aimed to determine whether the rs10748643 SNP corresponded to increased proportions of CD39 Tregs in an Australian donor population, and whether this SNP influences clinical GVHD in a humanized mouse model. Donors were genotyped for the rs10748643 SNP by Sanger sequencing, and the proportion of CD39 T cells in donor peripheral blood was determined by flow cytometry. Donors encoding the G allele (donors ) demonstrated higher proportions of CD39 CD3 CD4 CD25 CD127 Tregs, but not CD39 CD3 CD8 T cells or CD39 CD3 CD4 conventional T cells, compared with donors homozygous for the A allele (donors ). NOD-SCID-IL2Rγ mice were injected with human peripheral blood mononuclear cells from either donors (hCD39 mice) or donors (hCD39 mice). hCD39 mice demonstrated significantly greater weight loss and GVHD clinical scores, and significantly reduced survival, compared with hCD39 mice. hCD39 mice showed significantly higher hCD4 :hCD8 T-cell ratios than hCD39 mice, but displayed similar proportions of CD3 hCD4 hCD25 hCD127 Tregs and hCD39 Tregs. However, the proportion of human Tregs corresponded to survival in hCD39 mice, but not in hCD39 mice. This study demonstrates that donors encoding the G allele show higher percentages of CD39 Tregs, but cause worsened GVHD in humanized mice compared with donors homozygous for the A allele.