Myofibroblasts, key effector cells in tissue fibrosis, are specialized contractile cells. Lung myofibroblast contraction induces integrin αvβ5-dependent latent transforming growth factor (TGF)-β1 activation suggests that myofibroblast contractility may be a driving force for the persistent myofibroblast differentiation observed in fibrotic lungs. Understanding the mechanisms that regulate fibroblast contraction and mechanotransduction will add new insights into the pathogenesis of lung fibrosis and may lead to new therapeutic approaches for treating fibrotic lung diseases. We and others previously demonstrated that lung fibroblast expression of Thy-1 prevents lung fibrosis. The mechanisms underlying the anti-fibrotic effect of Thy-1 are not well understood. In this study, we showed that Thy-1 interacts with integrin αvβ5, both in a cell-free system and on the cell surface of rat lung fibroblasts. Thy-1-integrin αvβ5 interactions are RLD-dependent because mutated Thy-1, in which RLD is replaced by RLE, loses the ability to bind the integrin. Furthermore, Thy-1 expression prevents fibroblast contraction-induced, integrin αvβ5-dependent latent TGF-β1 activation and TGF-β1-dependent lung myofibroblast differentiation. In contrast, lack of Thy-1 expression or disruption of Thy-1-αvβ5 interactions renders lung fibroblasts susceptible to contraction-induced latent TGF-β1 activation and myofibroblast differentiation. These data suggest that Thy-1-integrin αvβ5 interactions inhibit contraction-induced latent TGF-β1 activation, presumably by blocking the binding of extracellular matrix-bound latent TGF-β1 with integrin α vβ5. Our studies suggest that targeting key mechanotransducers to inhibit mechanotransduction might be an effective approach to inhibit the deleterious effects of myofibroblast contraction on lung fibrogenesis. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.