Endothelial mechanotransduction by fluid shear stress (FSS) modulates endothelial function and vascular pathophysiology through mechanosensors on the cell membrane. The coxsackievirus and adenovirus receptor (CAR) is not only a viral receptor but also a component of tight junctions and plays an important role in tissue homeostasis. Here, we demonstrate the expression, regulatory mechanism, and role of CAR in vascular endothelial cells (ECs) under FSS conditions. Disturbed flow increased, whereas unidirectional laminar shear stress (LSS) decreased, CAR expression in ECs through the Krüppel-like factor 2 (KLF2)/activator protein 1 (AP-1) axis. Deletion of CAR reduced the expression of proinflammatory genes and endothelial inflammation induced by disturbed flow via the suppression of NF-κB activation. Consistently, disturbed flow-induced atherosclerosis was reduced in EC-specific CAR KO mice. CAR was found to be involved in endothelial mechanotransduction through the regulation of platelet endothelial cell adhesion molecule 1 (PECAM-1) phosphorylation. Our results demonstrate that endothelial CAR is regulated by FSS and that this regulated CAR acts as an important modulator of endothelial mechanotransduction by FSS.