Nitric oxide production in response to flow-dependent shear forces applied on the surface of endothelial cells is a fundamental mechanism of regulation of vascular tone, peripheral resistance, and tissue perfusion. This implicates the concerted action of multiple upstream “mechanosensing” molecules reversibly assembled in signalosomes recruiting endothelial nitric oxide synthase (eNOS) in specific subcellular locales, e.g., plasmalemmal caveolae. Subsequent short- and long-term increases in activity and expression of eNOS translate this mechanical stimulus into enhanced NO production and bioactivity through a complex transcriptional and posttranslational regulation of the enzyme, including by shear-stress responsive transcription factors, oxidant stress-dependent regulation of transcript stability, eNOS regulatory phosphorylations, and protein-protein interactions. Notably, eNOS expressed in cardiac myocytes is amenable to a similar regulation in response to stretching of cardiac muscle cells and in part mediates the length-dependent increase in cardiac contraction force. In addition to short-term regulation of contractile tone, eNOS mediates key aspects of cardiac and vascular remodeling, e.g., by orchestrating the mobilization, recruitment, migration, and differentiation of cardiac and vascular progenitor cells, in part by regulating the stabilization and transcriptional activity of hypoxia inducible factor in normoxia and hypoxia. The continuum of the influence of eNOS in cardiovascular biology explains its growing implication in mechanosensitive aspects of integrated physiology, such as the control of blood pressure variability or the modulation of cardiac remodeling in situations of hemodynamic overload.