Inhibition of the renin-angiotensin system prevents vascular dysfunction induced by estrogen deficiency in rats. However, the role of aldosterone in ovarian hormone deficiency-related vascular dysfunction is unclear. Therefore, we aimed to investigate the role of the aldosterone pathway in the endothelial dysfunction observed in isolated resistance and conduit arteries in a model of endogenous female hormone deficiency. Female Wistar rats (8 weeks old) underwent bilateral ovariectomy (Ovx) and were randomly assigned to receive daily treatment with spironolactone (Ovx Spi, 80 mg/kg), placebo (Ovx) and Sham treatment with spironolactone (Sham Spi, 80 mg/kg) or placebo (Sham group) for 60 days. In isolated aortic rings, but not mesenteric resistance arteries, Ovx increased vascular reactivity to phenylephrine that was prevented by spironolactone. Incubation with L-NAME increased the phenylephrine response in the isolated aorta in all groups, but this effect was smaller in Ovx rats. The muted response in the Ovx rats was restored by spironolactone. Apocynin, catalase, SOD, tiron and ML-171 attenuated the vascular reactivity to phenylephrine in the aorta of Ovx rats, but this effect was prevented by spironolactone. Corroborating these findings, the reduction of nitric oxide, and the increases in superoxide anion, hydrogen peroxide, NOX4 and NOX2 protein expression in aorta of Ovx rats were prevented by spironolactone. Therefore, spironolactone treatment prevented endothelial dysfunction in aorta from Ovx rats by increasing nitric oxide bioavailability and reducing NADPH oxidase-derived ROS production, suggesting a potential role of the pathway in the vascular dysfunction produced by female hormone deficiency in rats.