Studies employing T cell receptor transgenic T cells have convincingly shown that selective delivery of non-self model antigens to DEC-205+ dendritic cells (DCs) in the steady-state can induce Foxp3-expressing CD4+ CD25+ regulatory T (Treg) cells from conventional CD4+ CD25-Foxp3-T cells. Although of considerable clinical interest, the concept of DC-targeted de novo generation of antigen-specific Treg cells has not yet been evaluated for self-antigens and self-reactive CD4+ T cells in the non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D). Here, we show in proof-of-principle experiments that targeting a mimotope peptide to the endocytic receptor DEC-205 on DCs in NOD mice induces efficient conversion of pancreatic β-cell-reactive BDC2. 5 CD4+ T cells into long-lived Foxp3+ Treg cells. Of note, conversion efficiency in normoglycemic and hyperglycemic mice with early diabetes onset was indistinguishable. While de novo generation of BDC2. 5 Treg cells did not interfere with disease progression, anti-DEC-205-mediated targeting of whole proinsulin in prediabetic NOD mice substantially reduced the incidence of diabetes. These results suggest that promoting antigen-specific Treg cells in vivo might be a feasible approach towards cellular therapy in T1D.