Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease culminating in pancreatic ß-cell destruction due to the induction of an exacerbated pro-inflammatory milieu consisting of reactive oxygen species (ROS), cytokines, and islet-infiltrating leukocytes. We recently demonstrated the importance of NADPH oxidase (NOX)-derived ROS synthesis on autoimmune diabetes, as Non-Obese Diabetic (NOD) mice unable to generate superoxide (NOD.Ncf1m1J) were highly T1D resistant partly due to dampened macrophage responses and diminished levels of innate immune pro-inflammatory cytokines and Type I interferons upon Toll-like receptor ligation. Therefore, we hypothesized that NOX-derived ROS synthesis is necessary for efficient pro-inflammatory M1 macrophage differentiation/developmentand that loss of superoxide in NOD.Ncf1m1J mice may contribute to dampened pro-inflammatory M1 and/or enhanced anti-inflammatory M2 macrophage responses. During spontaneous T1D progression, islet-resident macrophages within female NOD.Ncf1m1J mice at 18 weeks of age exhibited an enhanced M2 macrophage phenotype in comparison to age-matched pre-diabetic NOD mice. We observed significant (p < 0.01) increases in mRNA accumulation of M2 macrophage markers, including Ccl17 (3-fold), Retnla (15-fold), Cd206 (2.5-fold), Arg1 (1.5-fold), and concomitant significant (p < 0.01) decreases in M1 markers, such as Cxcl10 (2-fold), Ccl5 (4-fold), Nos2 (8-fold), Ifng (8-fold), and Tnfa (8-fold). Adoptive transfer of diabetogenic BDC-2.5 splenocytes into NOD.Rag.Ncf1m1J mice resulted in 4- and 20-fold decreases in islet-infiltrating TNF-α+-expressing F4/80+ macrophages at 6 and 13 days post-transfer, respectively, in contrast to NOX-sufficient NOD.Rag mice, indicating an essential requirement for superoxide in pro-inflammatory M1 macrophage effector responses. Interestingly, M1-polarized NOD.Ncf1m1J bone marrow-derived macrophages exhibited a 2-fold increase in F4/80+/Arginase-1+ cells, indicative of an increased M2 macrophage phenotype, suggesting that superoxide has a role in macrophage plasticity and terminal differentiation. Our results demonstrate that ROS synthesis can influence pro-inflammatory M1 macrophage responses and immunotherapies targeting redox-dependent cues involved in innate immune responses may halt T1D progression.