Antigen-specific Tregs possess the potential to reduce excess immune responses in autoimmune diseases, allergy and organ transplantation. Although in vitro-expanded antigen-specific iTregs have been thought to be suitable for Treg therapy, iTregs are unstable compared with thymus-derived Tregs mostly due to methylation of the CpG islands of conserved noncoding sequence 2 (CNS2) region of the Foxp3 locus. In this study, we searched the optimum conditions to generate stable iTregs for the prevention of acute graft versus host disease (GVHD) model in mice. Alloantigen-specific iTregs were generated by co-culturing naive T cells with allogenic dendritic cells in the presence of TGF-β and retinoic acid. Among various agents and genes, we found that vitamin C most effectively retained Foxp3 expression in adoptively transferred iTregs in the GVDH model mice. As reported, vitamin C potentiated DNA demethylation of the Foxp3 CNS2 in alloantigen-specific iTregs. Adoptive transfer of vitamin C-treated iTregs ameliorated GVHD in mice more efficiently than untreated iTregs. These results strongly supports the utility of vitamin C in Treg therapy.