This study describes a robust method to expand antigen-specific regulatory T cells (Treg) from autoimmune-prone nonobese diabetic (NOD) mice. The expanded Treg were expanded up to 200-fold in less than 2 weeks using a combination of anti-CD3, anti-CD28, and interleukin 2. These expanded Treg retained their classical cell surface phenotype and functioned both in vitro and in vivo to suppress effector T cell functions. Significantly, small numbers of antigen-specific Treg could reverse diabetes after disease onset, suggesting a novel approach to cellular immunotherapy for autoimmunity. The results highlight the potential of antigen-specific Treg for treating autoimmune diseases and the importance of antigen specificity in their therapeutic efficacy.This study describes a robust method to expand antigen-specific regulatory T cells (Treg) from autoimmune-prone nonobese diabetic (NOD) mice. The expanded Treg were expanded up to 200-fold in less than 2 weeks using a combination of anti-CD3, anti-CD28, and interleukin 2. These expanded Treg retained their classical cell surface phenotype and functioned both in vitro and in vivo to suppress effector T cell functions. Significantly, small numbers of antigen-specific Treg could reverse diabetes after disease onset, suggesting a novel approach to cellular immunotherapy for autoimmunity. The results highlight the potential of antigen-specific Treg for treating autoimmune diseases and the importance of antigen specificity in their therapeutic efficacy.