Cardiac fibrosis is the excess deposition of myocardial extracellular matrix components, which increases tissue stiffness and heterogeneity, causing impaired diastolic/systolic function and arrhythmias, and eventually leading to heart failure and death. There are no available treatments for cardiac fibrosis. Myofibroblasts mediate fibrosis, and are characterized by hypersynthesis of collagens, decreased migration, and increased α-smooth muscle actin, which is incorporated into stress fibers, imparting contractility. Scleraxis is a transcriptional regulator of collagen-rich tissues, increased in response to the same stimuli that drive the myofibroblast phenotype, such as cyclic stretch. We show that Scleraxis mediates the conversion of cardiac fibroblasts to myofibroblasts, by increasing myofibroblast marker expression and contraction, and decreasing migration. Additionally, a proximal 1500 bp human SCLERAXIS promoter is activated by stretch and is responsive to transforming growth factor-β1. Thus, Scleraxis is a specific mechanoresponsive regulator of the myofibroblast, representing a novel target for the treatment of cardiac fibrosis.