The pathogenesis of cardiac valve disease correlates with the emergence of muscle-like fibroblasts (myofibroblasts). These cells display prominent stress fibers containing α-smooth muscle actin (α-SMA) and are believed to differentiate from valvular interstitial cells (VICs). However, the biological factors that initiate myofibroblast differentiation and activation in valves remain unidentified. We show that transforming growth factor-β1 (TGF-β1) mediates differentiation of VICs into active myofibroblasts in vitro in a dose-dependent manner, as determined by a significant increase in α-SMA and the dramatic augmentation of stress fiber formation and alignment. Additionally, TGF-β1 and increased mechanical stress function synergistically to enhance contractility. In turn, contractile valve myofibroblasts exert tension on the extracellular matrix, resulting in a dramatic realignment of extracellular fibronectin fibrils. TGF-β1 also inhibits valve myofibroblast proliferation without enhancing apoptosis. Our results are consistent with activation of a highly contractile myofibroblast phenotype by TGF-β1 and are the first to connect valve myofibroblast contractility with pathological valve matrix remodeling. We suggest that the activation of contractile myofibroblasts by TGF-β1 may be a significant first step in promoting alterations to the valve matrix architecture that are evident in valvular heart disease.