ART alters glucose homeostasis in mice and humans, but the underlying mechanisms are incompletely understood. ART induces endothelial dysfunction and arterial hypertension by epigenetic alteration of the eNOS gene. In eNOS deficient mice, insulin resistance is related to impaired insulin stimulation of muscle blood flow and substrate delivery and defective intrinsic skeletal muscle glucose uptake. We, therefore, assessed glucose tolerance, insulin sensitivity (euglycemic clamp), insulin stimulation of muscle blood flow in vivo and muscle glucose uptake in vitro in male ART and control mice fed a normal chow (NC) or challenged with a high fat diet (HFD) during 8 weeks. Glucose tolerance and insulin-sensitivity were similar in NC-fed animals. When challenged with HFD, however, ART mice developed exaggerated obesity, fasting hyperinsulinemia and hyperglycemia and a 20% lower insulin-stimulated glucose utilization than control mice (steady state GIR, 51.3±7.3 vs. 64.0±10.8 mg/kg • min, P=0.012). ART-induced insulin resistance was associated with defective insulin stimulation of muscle blood flow, whereas intrinsic skeletal muscle glucose uptake was normal. In conclusion, ART-induced endothelial dysfunction, when challenged with a metabolic stress facilitates glucose intolerance and insulin-resistance. Similar mechanisms may contribute to ART-induced alterations of the metabolic phenotype in humans.