CD4+ CD25+ Foxp3+ regulatory T cells (T reg) are critical for controlling autoimmunity. Evidence suggests that T reg development, peripheral maintenance, and suppressive function are dependent on Ag specificity. However, there is little direct evidence that the T reg responsible for controlling autoimmunity in NOD mice or other natural settings are Ag specific. In fact, some investigators have argued that polyclonal Ag-nonspecific T reg are efficient regulators of immunity. Thus, the goal of this study was to identify, expand, and characterize islet Ag-specific T reg in NOD mice. Ag-specific T reg from NOD mice were efficiently expanded in vitro using IL-2 and beads coated with recombinant islet peptide mimic-MHC class II and anti-CD28 mAb. The expanded Ag-specific T reg expressed prototypic surface markers and cytokines. Although activated in an Ag-specific fashion, the expanded T reg were capable of bystander suppression both in vitro and in vivo. Importantly, the islet peptide mimic-specific T reg were more efficient than polyclonal T reg in suppressing autoimmune diabetes. These results provide a direct demonstration of the presence of autoantigen-specific T reg in the natural setting that can be applied as therapeutics for organ-specific autoimmunity.