Type 1 diabetes (T1D) in both humans and nonobese diabetic (NOD) mice is a T cell-mediated autoimmune disease characterized by lymphocytic infiltration of pancreatic islets with subsequent destruction of the insulin-producing cells. The T regulatory (Treg) cell has been suggested to play an important role in controlling T cell-mediated inflammatory T1D. We previously demonstrated that induction of antigen-specific Treg cells in vivo by co-immunization with a DNA vaccine and its encoded protein can effectively inhibit T cell-mediated inflammatory diseases. To further demonstrate the potential of this strategy, we show here that co-immunization of NOD mice twice with DNA encoding proinsulin plus insulin protein prevents the onset of T1D and induces the impairment of antigen-specific T cell responses in a dose-dependent manner. We further show that the inhibitory function is due to the induction of TGF-beta-producing CD4(+)CD25(-) islet-specific iTreg cells against the onset of T1D in NOD mice. Induced iTreg cells were observed only in the co-immunization group, but derived neither from the DNA vaccine nor the protein alone, suggesting that a biased helper T cell type 1 response plays no inhibitory role. A strategy based on co-immunization to induce a protective response against the onset of diabetes in NOD mice may lead to the development of an immunotherapeutic/preventive protocol against T1D in humans.