Acute Graft-versus-Host Disease (aGvHD) is a severe and often life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), caused by donor T cells attacking recipient tissues. Clinical data suggest that acute myeloid leukemia (AML) patients receiving transplants from KIR3DS1+ donors exhibit lower aGvHD incidence and improved survival, though the underlying mechanisms remain unclear. KIR3DS1, an activating receptor on natural killer (NK) cells, is present in ~40% of the population. We demonstrate that prolonged T cell activation in vitro induces expression of HLA-F, a ligand for KIR3DS1, which promotes KIR3DS1+ NK cell degranulation. Using a xenogeneic aGvHD model in which human primary T and NK cells were transferred into NSG mice, we observed HLA-F upregulation on human T cells coinciding with clinical manifestations of aGvHD, such as weight loss and hunching. Notably, mice transplanted with cells from KIR3DS1+ donors or NK cells transduced to express KIR3DS1 showed reduced aGvHD severity, delayed onset, and improved survival compared to those receiving cells from KIR3DS1- donors. This protection was associated with slower T cell expansion, decreased infiltration of T cells in aGvHD target tissues (e.g., skin and intestine), and lower HLA-F expression on tissue-infiltrating T cells, suggesting that KIR3DS1+ NK cells target activated T cells or prevent their infiltration. Supporting these findings, a retrospective analysis of allo-HSCT patients revealed that individuals with aGvHD at day 100 post-transplantation had elevated CD8+ T cells with higher HLA-F expression by day 30. In summary, our findings reveal a novel KIR3DS1-mediated mechanism of T cell regulation in aGvHD and highlight the potential of selecting KIR3DS1+ donors for allo-HSCT to mitigate aGvHD risk and improve outcomes.