Myofibroblasts, specialized cells that play important roles in wound healing and fibrosis, can develop from epithelial cells through an epithelial-mesenchymal transition (EMT). During EMT, epithelial cells detach from neighboring cells and acquire an elongated, mesenchymal-like morphology. These phenotypic changes are accompanied by changes in gene expression patterns including upregulation of a variety of cytoskeletal associated proteins which contribute to the ability of myofibroblasts to exert large contractile forces. Here, the relationship between cell shape and cytoskeletal tension and the expression of cytoskeletal proteins in transforming growth factor (TGF)-β1-induced EMT is determined. We find that culturing cells in conditions which permit cell spreading and increased contractility promotes the increased expression of myofibroblast markers and cytoskeletal associated proteins. In contrast, blocking cell spreading prevents transdifferentiation to the myofibroblast phenotype. Furthermore, we find that cell shape regulates the expression of cytoskeletal proteins by controlling the subcellular localization of myocardin related transcription factor (MRTF)-A. Pharmacological inhibition of cytoskeletal tension or MRTF-A signaling blocks the acquisition of a myofibroblast phenotype in spread cells while overexpression of MRTF-A promotes the expression of cytoskeletal proteins for all cell shapes. These data suggest that cell shape is a critical determinant of myofibroblast development from epithelial cells.