Damage to the gastrointestinal tract following allogeneic hematopoietic stem cell transplantation is a significant contributor to the severity and perpetuation of graft-versus-host disease. In preclinical models and clinical trials, we showed that infusing high numbers of regulatory T-cells reduces graft-versus-host disease incidence. Despite no change in in vitro suppressive function, transfer of ex vivo expanded regulatory T-cells transduced to overexpress G-protein coupled receptor 15 or C-C Motif Chemokine Receptor 9, specific homing receptors for colon or small intestine, respectively, lessened graft-versus-host disease severity in mice. Increased regulatory T-cell frequency and retention within the gastrointestinal tissues of mice that received gut homing T-cells correlated with lower inflammation and gut damage early post-transplant, decreased graft-versus-host disease severity and prolonged survival compared to those receiving control transduced regulatory T-cells. These data provide evidence that enforced targeting of ex vivo expanded regulatory T-cells to the gastrointestinal tract diminishes gut injury and is associated with decreased graft-versus-host disease severity.