With the goal of identifying changes in gene expression in CD4⁺ T cells during the development of diabetes in the nonobese diabetic (NOD) mouse, we used DNA microarrays to analyze gene expression in CD4⁺ T cells from the pancreatic draining lymph nodes of NOD/BDC 2.5 T cell receptor transgenic and WT NOD mice at different ages. At 4 and 6 weeks of age, we found up-regulation of a number of genes that are known to be induced by IFN-α. IFN-α levels and IFN-α–producing plasmacytoid dendritic cells were increased in the PLNs of 3- to 4-week-old NOD mice. Moreover, blockade of IFN-α receptor 1 in NOD mice by a neutralizing antibody at 2–3 weeks of age significantly delayed the onset and decreased the incidence of type 1 diabetes, increased the relative number of immature dendritic cells in the PLNs, and enhanced the ability of spleen CD4⁺ T cells to produce IL-4 and IL-10. These findings demonstrate that IFN-α in the PLNs is an essential initiator in the pathogenesis of type 1 diabetes in NOD mice.