There is a significant body of research that demonstrates an association between central (visceral) obesity, insulin resistance, endothelial dysfunction and accelerated atherosclerosis, but the molecular mechanisms underlying this link are incompletely understood. In cultured human aortic endothelial cell (HAEC) models, insulin stimulates an intracellular signalling cascade resulting in activating phosphorylation of Insulin Receptor Substrate-1, Protein kinase B (PKB/Akt) and endothelial Nitric Oxide Synthase (eNOS), with subsequent NO release. Impairment of insulin stimulated nitric oxide (NO) production in the vascular endothelium is proposed to be a key mechanism in endothelial dysfunction. Visceral adiposity is associated with the disruption of normal physiological homeostasis of biologically active molecules secreted from adipose tissue (adipokines), with an increase in pro-inflammatory adipokines (e.g. Tumour Necrosis Factor Alpha, TNF-α). Adipokines are proposed to provide a key link between adiposity and the vasculature. Previous research in bovine tissue has demonstrated impairment of insulin-stimulated NO production by TNF-α, with impairment of the intracellular signalling cascade proposed as the mechanism of action. Accordingly, we have studied the interaction between TNF-α and insulin-mediated NO production in HAECs. We have demonstrated that pre-incubation with TNF-α inhibits insulin stimulated NO production. In contrast to bovine studies this was not associated with reduced activating phosphorylation of PKB (Ser473) or eNOS (Ser1177). The insulin stimulated phosphorylation state of previously uncharacterised eNOS residues has been determined (eNOS Ser114, 615 and 633), and these sites are also unaltered by TNF-α, implying that the endothelial signalling cascade remains intact. TNF-α induces superoxide production through activation of the NADPH oxidase system in our model. We propose that the reduction in insulin-stimulated NO seen with preincubation with TNF-α is not due to impairment of the insulin stimulated signalling cascade but is through superoxide production and quenching of NO, reducing overall NO bioavailability.