Graft-versus-host disease (GVHD) is the most lethal complication after allogeneic bone marrow transplantation (allo-BMT). Current approaches to prevent GVHD rely on donor lymphocyte/T cell depletion or general immunosuppression, leading to opportunistic infections and cancer relapse. Tolerogenic dendritic cells can induce regulatory T cells (Tregs) with the ability to suppress inflammation and prevent transplant rejection, making them an attractive cellular therapy to control GVHD. Active vitamin D (1α,25-dihydroxyvitamin D3; 1α,25(OH)2D3) promotes the generation of tolerogenic dendritic cells (1,25D3-DCs). This study aimed to determine the ability of ex vivo generated 1,25D3-DCs to trigger the expansion of Tregs that are able to control lethal xenogeneic GVHD in humanized NOD/LtSz-PrkdcscidIL2rγtm1Wjl (NSG) mice. We demonstrate that 1,25D3-DCs express lower levels of HLA-DR and costimulatory molecules, such as CD80 and CD86, and produce higher levels of IL-10 and TNF-α and lower amounts of IL-12, compared to vehicle-treated DCs. Moreover, these cells express increased levels of various co-inhibitory molecules such as PD-L1 and ILT-3 and the glycoprotein CD52 that is known to suppress T cell activation. Consequently, 1,25D3-DCs are poor stimulators of alloantigen-primed T cells, but foster the generation of antigen-specific suppressive Tregs. When adoptively transferred in humanized NSG mice, these 1,25D3-DC-induced Tregs delayed GVHD caused by the co-transferred autologous human peripheral blood mononuclear cells (PBMCs). These results indicate that 1,25D3-DC-induced Tregs can inhibit xenogeneic GVHD and maintain their immunomodulatory function under conditions of inflammation.