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 beta-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.