The biologically active form of vitamin D, 1,25-dihydroxyvitamin D ₃ [1,25(OH)₂D₃], is able to promote the generation of tolerogenic mature dendritic cells (mDCs) with an impaired ability to activate autoreactive T cells. These cells could represent a reliable tool for the promotion or restoration of Ag-specific tolerance through vaccination strategies, for example in type 1 diabetes patients. However, successful transfer of 1,25(OH)₂D₃-treated mDCs (1,25D ₃-mDCs) depends on the capacity of 1,25(OH)₂D₃ to imprint a similar tolerogenic profile in cells derived from diabetes-prone donors as from diabetes-resistant donors. In this study, we examined the impact of 1,25(OH)₂D₃ on the function and phenotype of mDCs originating from healthy (C57BL/6) and diabetes-prone (NOD) mice. We show that 1,25(OH)₂D₃ is able to imprint a phenotypic tolerogenic profile on DCs derived from both mouse strains. Both NOD- and C57BL/6-derived 1,25D₃-mDCs decreased the proliferation and activation of autoreactive T cells in vitro, despite strain differences in the regulation of cytokine/chemokine expression. In addition, 1,25D₃-mDCs from diabetes-prone mice expanded CD25+Foxp3+ regulatory T cells and induced intracellular IL-10 production by T cells in vitro. Furthermore, 1,25D ₃-mDCs exhibited an intact functional migratory capacity in vivo that favors homing to the liver and pancreas of adult NOD mice. More importantly, when cotransferred with activated CD4+ T cells into NOD.SCID recipients, 1,25D₃-mDCs potently dampened the proliferation of autoreactive donor T cells in the pancreatic draining lymph nodes. Altogether, these results argue for the potential of 1,25D₃-mDCs to restore Ag-specific immune tolerance and arrest autoimmune disease progression in vivo. The Journal of Immunology, 2014, 192: 4210-4220.