Vascular cells respond to supraphysiological amounts of stretch with a characteristic phenotypic change that results in dysfunctional remodeling of the affected arteries. Although the pathophysiological consequences of stretch-induced signaling are well characterized, the mechanism of mechanotransduction is unclear. We focused on the mechanotransducer zyxin, which translocates to the nucleus to drive gene expression in response to stretch. In cultured human endothelial cells and perfused femoral arteries isolated from wild-type and several knockout mouse strains, we characterized a multistep signaling pathway whereby stretch led to a transient receptor potential channel 3-mediated release of the endothelial vasoconstrictor peptide endothelin-1 (ET-1). ET-1, through autocrine activation of its B-type receptor, elicited the release of pro-atrial natriuretic peptide (ANP), which caused the autocrine activation of the ANP receptor guanylyl cyclase A (GC-A). Activation of GC-A, in turn, led to protein kinase G-mediated phosphorylation of zyxin at serine 142, thereby triggering the translocation of zyxin to the nucleus, where it was required for stretch-induced gene expression. Thus, we have identified a stretch-induced signaling pathway in vascular cells that leads to the activation of zyxin, a cytoskeletal protein specifically involved in transducing mechanical stimuli.