Type 1 diabetes in NOD mice is characterized by the uncontrolled Th1 immune responses and deficiency of regulatory or suppressor cells. Previous study has shown that NOD mice treated with polyinosinic-polycytidylic acid (poly(I:C)) have a markedly reduced incidence of diabetes, but the underlying mechanisms remain unclear. In this study, we report that the prevention of diabetes by poly(I:C) is associated with the formation of Th2-enriched environment in spleen and pancreas. We further show that the prevention of diabetes and the formation of Th2-enriched environment depend on the presence of NK cells. Long-term poly(I:C)-treated NK cells exhibit a NK3-like phenotype, and are involved in the induction of Th2 bias of spleen cells in response to islet autoantigens via TGF-beta-dependent manner. Therefore, NK cells mediate the protective effect of poly(I:C) possibly through the promotion of Th2 bias of immune responses. These findings suggest that NK cells can participate in the regulation of autoimmune diabetes.