In type 1 diabetes (T1D), reactive oxygen species (ROS) and proinflammatory cytokines produced by macrophages and other innate immune cells destroy pancreatic β cells while promoting autoreactive T cell maturation. Superoxide-deficient nonobese diabetic mice (NOD.Ncf1 m1J) are resistant to spontaneous diabetes, revealing the integral role of ROS signaling in T1D. Here, we evaluate the innate immune activation state of bone marrow-derived macrophages (BM-Mφ) from NOD and NOD.Ncf1 m1J mice after poly(I:C)-induced Toll-like receptor 3 (TLR3) signaling. We show that ROS synthesis is required for efficient activation of the NF-κB signaling pathway and concomitant expression of TLR3 and the cognate adaptor molecule, TRIF. Poly(I:C)-stimulated NOD.Ncf1 m1J BM-Mφ exhibited a 2- and 10-fold decrease in TNF-α and IFN-β proinflammatory cytokine synthesis, respectively, in contrast to NOD BM-Mφ. Optimal expression of IFN-α/β is not solely dependent on superoxide synthesis, but requires p47 phox to function in a NOX-independent manner to mediate type I interferon synthesis. Interestingly, MHC-II I-A g7 expression necessary for CD4 T cell activation is increased 2-fold relative to NOD, implicating a role for superoxide in I-A g7 downregulation. These findings suggest that defective innate immune-pattern-recognition receptor activation and subsequent decrease in TNF-α and IFN-β proinflammatory cytokine synthesis necessary for autoreactive T cell maturation may contribute to the T1D protection observed in NOD.Ncf1 m1J mice. © 2012 Elsevier Inc. All rights reserved.