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^AG/GG) demonstrated higher proportions of CD39⁺CD3⁺CD4⁺CD25⁺CD127^lo Tregs, but not CD39⁺CD3⁺CD8⁺ T cells or CD39⁺CD3⁺CD4⁺ conventional T cells, compared with donors homozygous for the A allele (donors^AA). NOD‐SCID‐IL2Rγ^null mice were injected with human peripheral blood mononuclear cells from either donors^AA (hCD39^AA mice) or donors^AG/GG (hCD39^AG/GG mice). hCD39^AG/GG mice demonstrated significantly greater weight loss and GVHD clinical scores, and significantly reduced survival, compared with hCD39^AA mice. hCD39^AG/GG mice showed significantly higher hCD4⁺:hCD8⁺ T‐cell ratios than hCD39^AA mice, but displayed similar proportions of CD3⁺hCD4⁺hCD25⁺hCD127^lo Tregs and hCD39⁺ Tregs. However, the proportion of human Tregs corresponded to survival in hCD39^AA mice, but not in hCD39^AG/GG 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.