Cardiovascular disease is a leading cause of death and a major economic burden in the developed and developing world. Many heart diseases, including post-myocardial infarction, include a fibrotic component with remodeling of the extracellular matrix in the myocardium. Cardiac myofibroblasts are non-myocyte cells derived from relatively quiescent fibroblasts and are the main mediators of collagen remodeling in disease states. TGF-β is recognized as an important contributor to adverse cardiac remodeling in heart disease. In this study we have investigated the role of c-Ski, which is an endogenous TGF-β inhibitor, in controlling/regulating myofibroblast function and phenotype. We have developed an adenoviral overexpression system to study these endpoints using Western blot, immunofluorescence, MTT assay, flow cytometry, procollagen type I amino terminal peptide secretion and qPCR analysis. We observed that the 95 kDa c-Ski form is overexpressed upon virus infection with adenovirus encoding c-Ski and this form of c-Ski is localized to the nucleus. c-Ski expression inhibited cardiac myofibroblast collagen synthesis and secretion as well as contractility. Phosphorylation and translocation of Smad2 into the nucleus was not affected in the presence of c-Ski overexpression. We found that c-Ski overexpression was associated with diminution of the myofibroblastic phenotype with reduced α-smooth muscle actin and extra domain-A fibronectin expression (but not non-muscle myosin heavy chain B expression). c-Ski may reduce cell viability via the induction of apoptosis. Finally, we have elucidated a putative mechanism for c-Ski-mediated reduction of myofibroblast phenotype through the upregulation of the homeodomain protein Meox2. Adenoviral overexpression of Meox2 was associated with a significant reduction of α-smooth muscle actin and extra domain-A fibronectin expression in a similar manner to that of c-Ski overexpression. Thus we have identified c-Ski as being an antifibrotic protein as well as a novel mechanism for modulation of cardiac myofibroblast phenotype, possibly through the induction of Meox2 expression.