DNA vaccination encoding β cell autoantigens has been shown very recently to prevent type I diabetes in non-obese diabetic (NOD) mice. However, DNA vaccination encoding microbial or reporter antigens is known to induce specific long-lasting CD4 Th1 and strong cytolytic CD8 T cell responses. As this immune phenotype is associated strongly with β cell destruction leading to diabetes, we have chosen to study the effects of plasmids encoding glutamic acid decarboxylase (GAD), a crucial β cell autoantigen, in female NOD mice that developed a ‘moderate’ diabetes incidence. In the present study, 3-week-old female NOD mice were vaccinated twice in tibialis muscles with plasmid-DNA encoding 65-kDa GAD or βgalactosidase. In GAD-DNA immunized mice, diabetes cumulative incidence (P < 3·10−3) and insulitis (P < 7·10−3) increased significantly. Simultaneously, DNA immunization induced GAD-specific CD4 T cells secreting interleukin (IL)-4 (P < 0·05) and transforming growth factor (TGF)-β (P = 0·03). These cells were detected in spleen and in pancreatic lymph nodes. Furthermore, vaccination produced high amounts of Th2 cytokine-related IgG1 (P < 3·10−3) and TGF-β-related IgG2b to GAD (P = 0·015). Surprisingly, diabetes onset was correlated positively with Th2-related GAD-specific IgG1 (P < 10−4) and TGF-β-related IgG2b (P < 3·10−3). Moreover, pancreatic lesions resembled Th2-related allergic inflammation. These results indicate, for the first time, that GAD-DNA vaccination could increase insulitis and diabetes in NOD mice. In addition, our study suggests that Th2/3 cells may have potentiated β cell injury.