Acute graft-versus-host disease (GVHD) limits the success of allogeneic hematopoietic cell transplantation (allo-HCT); therefore, a better understanding of its biology may improve therapeutic options. We observed miR-146a up-regulation in T cells of mice developing acute GVHD compared with untreated mice. Transplanting miR-146a^−/− T cells caused increased GVHD severity, elevated tumor necrosis factor (TNF) serum levels, and reduced survival. TNF receptor-associated factor 6 (TRAF6), a verified target of miR-146a, was up-regulated in miR-146a^−/− T cells following alloantigen stimulation. Higher TRAF6 levels translated into increased nuclear factor-κB activity and TNF production in miR-146a^−/− T cells. Conversely, the detrimental effect of miR-146a deficiency in T cells was antagonized by TNF blockade, whereas phytochemical induction of miR-146a or its overexpression using a miR-146a mimic reduced GVHD severity. In humans, the minor genotype of the single nucleotide polymorphism rs2910164 in HCT donors, which reduces expression of miR-146a, was associated with severe acute GVHD (grade III/IV). We show that miR-146a functions as a negative regulator of donor T cells in GVHD by targeting TRAF6, leading to reduced TNF transcription. Because miR-146a expression can be exogenously enhanced, our results provide a novel targeted molecular approach to mitigate GVHD.