Methylglyoxal (MGO) is a metabolite of glucose and likely related to pathogenesis of diabetes-related vascular complications including hypertension. In this study, long-term effects of MGO on endothelial function were examined. Rat isolated mesenteric artery was treated for 3 days with MGO using an organ culture method. The contractility, morphology and protein expression of organ-cultured artery were examined. MGO (42 μM, 3 days) impaired acetylcholine (ACh: 1 nM–300 μM)-induced endothelium-dependent relaxation, while it had no effect on sodium nitroprusside (0.1 nM–10 μM)-induced endothelium-independent relaxation. MGO decreased ACh (3 μM)-induced nitric oxide (NO) production as measured by a fluorescence NO indicator, diaminofluorescein-2. Consistently, MGO inhibited ACh (3 μM)-induced phosphorylation of vasodilator stimulated phosphoprotein (an indicator of cyclic GMP production). MGO induced apoptosis in endothelium as detected by TdT-mediated dUTP-biotin nick-end labeling staining. MGO induced accumulation of superoxide in endothelium as detected by dihydroethidium staining. MGO decreased protein expression of endothelial NO synthase (eNOS). Gp91ds-tat (0.1 μM), an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), prevented the impairment of endothelium-dependent relaxation and the decrease in eNOS protein caused by MGO. The present results demonstrated that long-term MGO treatment impairs endothelium-dependent relaxation through NOX-derived increased superoxide-mediated endothelial apoptosis.