Innate immunity is mediated, at least in part, through a number of receptors known as Toll-like receptors (TLRs), which are activated by different microbial stimuli. Adaptive immunity, including autoimmunity, follows the innate response in a more specific manner. To investigate the roles of TLR3 and TLR9 in the development of type 1 diabetes, we generated NOD mice that are deficient in TLR3 and 9, respectively. There was no obvious difference in the incidence of spontaneous diabetes between TLR3-deficient mice and TLR3 heterozygous mice. However, TLR9-deficient mice were markedly protected from the disease compared to TLR9 heterozygous mice. Our results suggest that different TLRs play a varying role in autoimmune diseases.