The prevailing dogma for the development of type 1 diabetes (T1D) involves the stepwise sequence of autoimmunity, β-cell destruction, insulin deficiency, and hyperglycemia, the chronic effects of which are accepted as the causative and exacerbating factors in diabetic vascular complications. The connection between glycemic control and attenuation of complications in extant T1D has been well established. Recently, however, our description of oxidant stress-mediated endothelial dysfunction in prediabetic, euglycemic nonobese diabetic (NOD) mice has disputed hyperglycemia as the instigating factor in diabetic vascular complications. In this translational study, we asked if serum from patients with anti-β-cell antibodies (prediabetic patients) was capable of increasing the rate of reactive oxygen species (ROS) production in vitro. Human umbilical vein endothelial cells were incubated for 1 hour with media containing serum from either prediabetic, diabetic, or control patients. Rate of intracellular ROS generation was determined via spectrofluorometric assessment of a fluorescein diacetate assay. ROS generation was higher in cells incubated with prediabetic serum compared to control serum (+76%). Surprisingly, incubation of cells with diabetic serum yielded little difference from control serum. ROS generation persisted when cells were incubated with the IgG fraction of prediabetic serum but was negligible with the non-IgG fraction. Furthermore, use of DPI, an inhibitor of NAD(P)H oxidase, decreased the rate of ROS generation in all samples. These data implicate IgG activation of a vascular NAD(P)H oxidase, leading to increased production of intracellular ROS in prediabetic patients, prior to extant diabetes. This correlates with our previous findings in prediabetic NOD mice, supporting the conclusion that oxidant stress mediated endothelial dysfunction occurs prior to glycemic dysregulation in a subset of patients.