Apoptosis of fibroblasts/myofibroblasts is a critical event in the resolution of tissue repair responses; however, mechanisms for the regulation of (myo)fibroblast apoptosis/survival remain unclear. In this study, we demonstrate counter-regulatory interactions between the plasminogen activation system and transforming growth factor-β1 (TGF-β1) in the control of fibroblast apoptosis. Plasmin treatment induced fibroblast apoptosis in a time- and dose-dependent manner in association with proteolytic degradation of extracellular matrix proteins, as detected by the release of soluble fibronectin peptides. Plasminogen, which was activated to plasmin by fibroblasts, also induced fibronectin proteolysis and fibroblast apoptosis, both of which were blocked by α2-antiplasmin but not by inhibition of matrix metalloproteinase activity. TGF-β1 protected fibroblasts from apoptosis induced by plasminogen but not from apoptosis induced by exogenous plasmin. The protection from plasminogen-induced apoptosis conferred by TGF-β1 is associated with the up-regulation of plasminogen activator-1 (PAI-1) expression and inhibition of plasminogen activation. Moreover, lung fibroblasts from mice genetically deficient in PAI-1 lose the protective effect of TGF-β1 against plasminogen-induced apoptosis. These findings support a novel role for the plasminogen activation system in the regulation of fibroblast apoptosis and a potential role of TGF-β1/PAI-1 in promoting (myo)fibroblast survival in chronic fibrotic disorders.