Studies of animals with spontaneous autoimmune diabetes have revealed that autoreactive T cells that mediate islet beta-cell destruction belong to the Th1 subset (producing IL-2 and IFN-gamma), whereas regulatory T cells are Th2 type (producing IL-4 and IL-10). Here, we evaluate the effect of combined delivery of plasmid DNA encoding IL-4 and IL-10 using a degradable, cationic polymeric carrier, poly[gamma-(4-aminobutyl)-L-glycolic acid] (PAGA), in nonobese diabetic (NOD) mice. In the liver of NOD mice, we detected mouse Il4 and Il10 mRNA 5 days after intravenous injection of both PAGA-Il4 and PAGA-Il10 plasmid complexes. We found that 6 weeks after injection, 75% of observed islets were intact compared with less than 3% in the control group. Furthermore, in the treatment group, only 5% of the islets were severely infiltrated by the lymphocytes compared with over 30% in the control group. We measured glucose levels weekly up to the age of 32 weeks, revealing that co-injection of PAGA-Il4 and PAGA-Il10 plasmids prevented the development of diabetes in 75% of the treated animals. Thus, combined administration of mouse Il4 and Il10 plasmids prevents the development of autoimmune diabetes in NOD mice.