The study investigates the mechanism by which myofibroblasts (Mfs) activate latent transforming growth factor β1 (TGF-β1) from the extracellular matrix (ECM) in response to mechanical stress. Mfs, which are responsible for tissue repair and fibrosis, are shown to release active TGF-β1 through contraction of their cytoskeleton. This process is independent of protease activity and is mediated by integrin binding to latency-associated protein (LAP) in the latent complex (LLC) of TGF-β1. The activation of latent TGF-β1 is enhanced by increasing ECM stiffness and is inhibited by reducing ECM compliance or antagonizing integrins. In vivo, mechanical stress applied to wounds increases the activation of TGF-β1 signaling targets, such as Smad2/3, in stressed compared to relaxed tissues. The findings suggest that the activation of TGF-β1 by Mf contraction may serve as a checkpoint in fibrosis progression, restricting autocrine TGF-β1 production to a stiffened ECM.The study investigates the mechanism by which myofibroblasts (Mfs) activate latent transforming growth factor β1 (TGF-β1) from the extracellular matrix (ECM) in response to mechanical stress. Mfs, which are responsible for tissue repair and fibrosis, are shown to release active TGF-β1 through contraction of their cytoskeleton. This process is independent of protease activity and is mediated by integrin binding to latency-associated protein (LAP) in the latent complex (LLC) of TGF-β1. The activation of latent TGF-β1 is enhanced by increasing ECM stiffness and is inhibited by reducing ECM compliance or antagonizing integrins. In vivo, mechanical stress applied to wounds increases the activation of TGF-β1 signaling targets, such as Smad2/3, in stressed compared to relaxed tissues. The findings suggest that the activation of TGF-β1 by Mf contraction may serve as a checkpoint in fibrosis progression, restricting autocrine TGF-β1 production to a stiffened ECM.