Epithelial-mesenchymal-myofibroblast transition (EMT), a key feature in organ fibrosis, is regulated by the state of intercellular contacts. Our recent studies have shown that an initial injury of cell–cell junctions is a prerequisite for transforming growth factor-β1 (TGF-β1)-induced transdifferentiation of kidney tubular cells into α-smooth muscle actin (SMA)–expressing myofibroblasts. Here we analyzed the underlying contact-dependent mechanisms. Ca²⁺ removal–induced disruption of intercellular junctions provoked Rho/Rho kinase (ROK)-mediated myosin light chain (MLC) phosphorylation and Rho/ROK-dependent SMA promoter activation. Importantly, myosin-based contractility itself played a causal role, because the myosin ATPase inhibitor blebbistatin or a nonphosphorylatable, dominant negative MLC (DN-MLC) abolished the contact disruption-triggered SMA promoter activation, eliminated the synergy between contact injury and TGF-β1, and suppressed SMA expression. To explore the responsible mechanisms, we investigated the localization of the main SMA-inducing transcription factors, serum response factor (SRF), and its coactivator myocardin-related transcription factor (MRTF). Contact injury enhanced nuclear accumulation of SRF and MRTF. These processes were inhibited by DN-Rho or DN-MLC. TGF-β1 strongly facilitated nuclear accumulation of MRTF in cells with reduced contacts but not in intact epithelia. DN-myocardin abrogated the Ca²⁺-removal– ± TGF-β1–induced promoter activation. These studies define a new mechanism whereby cell contacts regulate epithelial-myofibroblast transition via Rho-ROK-phospho-MLC–dependent nuclear accumulation of MRTF.