Enhanced activation of the endothelial mineralocorticoid receptor contributes to the development of arterial stiffness, which is an independent predictor of cardiovascular disease. Previously we showed that enhanced endothelium mineralocorticoid receptor signaling in female mice prompts expression and translocation of the alpha subunit of the epithelial sodium channel (ENaC) to the endothelial cell (EC) surface (EnNaC) inducing vascular fibrosis and stiffness. Further, amiloride, an ENaC antagonist, inhibits vascular fibrosis, remodeling, and stiffness induced by feeding a Western diet high in saturated fat and refined carbohydrates. However, how this occurs remains unknown. Thereby, we hypothesized that EC-specific EnNaC activation is necessary for aldosterone mediated endothelium stiffness. To address this notion EnNaC α subunit knock out (EnNaC −/−) and wild type littermate female mice were administrated aldosterone (250 μg/kg/day) via osmotic minipumps for 3 weeks beginning at 25–28 weeks of age. In isolated mouse ECs, inward sodium currents were significantly reduced in amiloride controls as well as in EnNaC −/−. Likewise, aldosterone-induced endothelium stiffness was increased and endothelium dependent relaxation less in EnNaC −/− versus wild type. Further, EnNaC −/− mice exhibited attenuated responses to aldosterone infusion including: aortic endoplasmic reticulum stress, endothelium nitric oxide synthase activation, endothelium permeability, expression of pro-inflammatory cytokines, oxidative stress, and aortic collagen 1 deposition, supporting the notion that αEnNaC subunit activation contributes to these vascular responses.