Adoptive transfer of CD4+ CD25+ regulatory T cells has been shown to have therapeutic effects in experimental graft-vs-host disease (GVHD) models. Chemokines play an important role in the recruitment of alloreactive donor T cells into target organs during GVHD. In this study, we investigated the effectiveness of targeted delivery of CD4+ CD25+ regulatory T cells via a transfected chemokine receptor on reduction of organ damage during acute GVHD. High levels of expression of Th1-associated chemokines (CXCL9, CXCL10 and CXCL11) and their receptor CXCR3 were observed in the liver, lung and intestine of GVHD-induced recipient mice. Recipient mice that had undergone transfer of CD4+ CD25+ Foxp3+ CXCR3-transfected T cells (CXCR3-Treg cells) showed significant amelioration of GVHD changes in the liver, lung and intestine in comparison with recipient mice that had received CD4+ CD25+ Foxp3+ T cells (Treg cells) or naturally occurring CD4+ CD25+ regulatory T cells. This was due to more pronounced migration of CXCR3-Treg cells and their localization for a longer time in Th1-associated chemokine-expressing organs, resulting in stronger suppressive activity. We succeeded in preparing chemokine receptor-expressing Treg cells and demonstrated their ability to ameliorate disease progression upon accumulation in target organs. This method may provide a new therapeutic approach for organ damage in acute GVHD.