The phenotype conversion of fibroblasts to myofibroblasts plays a key role in the pathogenesis of cardiac fibrosis. Numerous triggers of this conversion process have been identified, including plating of cells on solid substrates, cytokines such as Transforming Growth Factor β, and mechanical stretch, however the underlying mechanisms remain incompletely defined. Recent studies from our laboratory revealed that the transcription factor scleraxis is a key regulator of cardiac fibroblast phenotype and extracellular matrix expression. Here we report that mechanical stretch induces type I collagen expression and morphological changes indicative of cardiac myofibroblast conversion, as well as scleraxis expression via activation of the scleraxis promoter. Scleraxis causes phenotypic changes similar to stretch, and the effect of stretch is attenuated in scleraxis null cells. Scleraxis was also sufficient to up-regulate expression of vinculin and F-actin, to induce stress fiber and focal adhesion formation, and to attenuate both cell migration and proliferation - further evidence of scleraxis-mediated regulation of fibroblast to myofibroblast conversion. Together, these data confirm that scleraxis is sufficient to promote the myofibroblast phenotype, and is a required effector of stretch-mediated conversion. Scleraxis may thus represent a potential target for the development of novel anti-fibrotic therapies aimed at inhibiting myofibroblast formation.