Insulin resistance is established as an independent risk factor for the development of type 2 diabetes and cardiovascular atherosclerosis. Most studies have examined atherogenesis in models of severe insulin resistance or diabetes. However, by the time of diagnosis, individuals with type 2 diabetes already demonstrate a significant atheroma burden. Furthermore, recent studies suggest that even in adolescence, insulin resistance increases cardiovascular risk. In the present report we studied early mechanisms of reduction in the bioavailability of the antiatheroscerotic molecule nitric oxide (NO) in very mild insulin resistance. Mice with haploinsufficiency for the insulin receptor (IRKO) represent a model of mild insulin resistance with preserved glycaemic control. 2 month-old IRKO mice have preserved vasorelaxation responses to acetylcholine (Ach). This remained the case at 4 months of age. However, by 6 months, despite no significant deterioration in glucose homeostasis, (Adult) IRKO mice had blunting of Ach-mediated vasorelaxation (IRKO Emax 66±5% versus WT 87±4%, p<0.01). Despite the endothelial dysfunction demonstrated, aortic eNOS mRNA levels were similar in Adult IRKO and WT mice, and interestingly, eNOS protein levels were increased suggesting a compensatory upregulation in the IRKO. The superoxide dismutase mimetic MnTMPyP restored Ach relaxation responses in the Adult IRKO (Emax to Ach with MnTMPyP 85±5%). Dihydroethidium fluorescence of aortae and isolated coronary microvascular endothelial cells confirmed a substantial increase in endothelium-derived reactive oxygen species in IRKO mice. Mild insulin resistance therefore represents a potent substrate for accelerated endothelial dysfunction. Furthermore, endothelial cell superoxide production mediates the early reduction in NO bioavailability.