Plasma adiponectin level is significantly reduced in patients with metabolic syndrome and vascular dysfunction is an important pathologic event in these patients. However, whether adiponectin may protect endothelial cells and attenuate endothelial dysfunction caused by metabolic disorders remains largely unknown. Adult rats were fed with a regular or a high-fat diet for 14 weeks. The aorta was isolated and vascular segments were incubated with vehicle or the globular domain of adiponectin (gAd, 2 µg/ml) for 4 h. The effect of gAd on endothelial function, nitric oxide (NO) and superoxide production, nitrotyrosine formation, gp91^phox expression, and eNOS/iNOS activity/expression was determined. Severe endothelial dysfunction (maximal vasorelaxation to ACh: 70.3±3.3% vs. 95.2±2.5% in control, P0.01) was observed in hyperlipidemic aortic segments and treatment with gAd significantly improved endothelial function (P0.01). Paradoxically, total NO production was significantly increased in hyperlipidemic vessels and treatment with gAd slightly reduced, rather than increased, total NO production in these vessels. Treatment with gAd reduced (-78%, P<0.01) superoxide production and peroxynitrite formation in hyperlipidemic vascular segments. Moreover, a moderate attenuation (-30%, P0.05) in gp91^phox and iNOS overexpression in hyperlipidemic vessels was observed after gAd incubation. Treatment with gAd had no effect on eNOS expression but significantly increased eNOS phosphorylation (P0.01). Most noticeably, treatment with gAd significantly enhanced eNOS (+83%) but reduced iNOS (-70%, P0.01) activity in hyperlipidemic vessels. Collectively, these results demonstrated that adiponectin protects the endothelium against hyperlipidemic injury by multiple mechanisms including promoting eNOS activity, inhibiting iNOS activity, preserving bioactive NO, and attenuating oxidative/nitrative stress.