An increase in reactive oxygen species (ROS) through NADPH oxidase activation frequently follows stress that activates beta-adrenoreceptors, leading to deterioration of cardiovascular disease. We hypothesized that upregulation of NADPH oxidase in the vasculature causes mild vascular spasm subsequent to chronic isoproterenol (ISO) administration, correlating significantly with activation of both ETA and ETB receptors. We tested whether the dual endothelin receptor antagonist CPU0213 is effective in reversing ISO-induced vascular abnormality by suppressing activated NADPH oxidase in the vasculature. Rats were injected with ISO (1 mg/kg, SC) for 10 days to induce vascular dysfunction and treated with CPU0213 (30 mg/kg, SC) or aminoguanidine (AMG, an inhibitor of iNOS, 100 mg/kg, PO) from day 7 to day 10. On day 11, we found an increase in vascular response to phenylephrine (Phe) and reductions in NO availability and acetylcholine (ACh)-induced relaxation in ISO-treated rats along with upregulated mRNA of ETA, ETB, iNOS, NADPH oxidase-Phox22 and Phox47, and matrix metalloproteinase 9 (MMP9). These abnormalities were attenuated by 3 days of intervention with CPU0213 but less with AMG. CPU0213 was more effective in relieving enhanced vascular constriction and reversal of ET receptor and MMP9 expression in the vasculature than was AMG. In conclusion, an upregulation of NADPH oxidase phox 22 and phox 47, ETA and ETB, and MMP9 correlates with vascular abnormality and the endothelin receptor antagonist CPU0213 was more effective than AMG in reversing ISO-induced enhanced vascular constriction by normalizing the above abnormal expression.