Diabetogenic CD8+ T cells are primed in the pancreatic lymph nodes (PLNs) by dendritic cells (DCs) carrying islet cell Ags. TLR signaling modifies DC function. The goal of this study was to determine the effect of TLR9 signaling on diabetogenic CD8+ T cell activation and the course of type 1 diabetes. We explored the effects of CpG oligonucleotide, TLR9 antagonists, and genetic TLR9 deficiency on the activation of diabetogenic CD8+ T cells. NOD bone marrow-derived DCs pulsed with freeze-thawed insulinoma cells in the presence of TLR9 agonist CpG and CD40 agonist induced diabetogenic CD8+ T cell activation. The addition of TLR9 antagonist oligodeoxynucleotide or chloroquine inhibited bone marrow-derived DCs activation and CD8+ T cell priming in response to CpG. CpG alone or with CD40 agonist induced CTL activity that triggered diabetes development in 8.3-TCR transgenic NOD mice. Oligodeoxynucleotide treatment of 8.3-TCR transgenic NOD mice delayed spontaneous diabetes development. Chloroquine treatment delayed the spontaneous onset of diabetes in NOD mice, coincident with the decreased activation of PLN DCs. TLR9−/− NOD mice had delayed onset of diabetes compared with TLR9−/+ NOD littermates. TLR9−/− NOD mice had lower levels of IFN-α in PLNs and decreased frequencies of plasmacytoid DCs and diabetogenic CD8+ T cells compared with NOD mice. We propose that TLR9 activation contributes to the spontaneous onset of diabetes in NOD mice by increasing IFN-α and promoting diabetogenic CD8 T cell activation.