Insulin has been reported as a major autoantigen in both human and murine type 1 diabetes (T1D). Insulin1-knockout NOD mice with only insulin2 are protected against the development of autoimmune diabetes, suggesting that insulin1 has strong immunogenicity and insulin2 has weak immunogenicity or a possible protective role in the pathogenesis of type 1 diabetes. In this study, we have developed fiber-mutant adenovirus vectors that express murine proinsulin1 or proinsulin2 (named Ad.Pins1-RGD/Ad.Pins2-RGD) and administered those virus vectors to the NOD mouse to evaluate modulation of autoimmune responses. The intravenous administration of either Ad.Pins1-RGD or Ad.Pins2-RGD at 3 and 5 weeks of age strongly suppressed the development of overt diabetes, accompanied by a significant reduction of insulin autoantibody (IAA), and suppression of disease was similar between administration of Ad.Pins1-RGD and that of Ad.Pins2-RGD. Our study suggests that systemic administration of fiber-mutant adenovirus vectors, which induce transient expression of proinsulin, may be applicable to a gene therapy inducing tolerance to insulin.