Vascular stiffness is a mechanical property of the vessel wall that affects blood pressure, permeability, and inflammation. As a result, vascular stiffness is a key driver of (chronic) human disorders, including pulmonary arterial hypertension, kidney disease, and atherosclerosis. Responses of the endothelium to stiffening involve integration of mechanical cues from various sources, including the extracellular matrix, smooth muscle cells, and the forces that derive from shear stress of blood. This response in turn affects endothelial cell contractility, which is an important property that regulates endothelial stiffness, permeability, and leukocyte–vessel wall interactions. Moreover, endothelial stiffening reduces nitric oxide production, which promotes smooth muscle cell contraction and vasoconstriction. In fact, vessel wall stiffening, and microcirculatory endothelial dysfunction, precedes hypertension and thus underlies the development of vascular disease. Here, we review the cross talk among vessel wall stiffening, endothelial contractility, and vascular disease, which is controlled by Rho-driven actomyosin contractility and cellular mechanotransduction. In addition to discussing the various inputs and relevant molecular events in the endothelium, we address which actomyosin-regulated changes at cell adhesion complexes are genetically associated with human cardiovascular disease. Finally, we discuss recent findings that broaden therapeutic options for targeting this important mechanical signaling pathway in vascular pathogenesis.