Transforming growth factor ₁ (TGF-₁) signal and downstream effectors Smads family play an important role in tissue fibrosis and matrix remodeling in various etiologies of heart failure. Inhibitory Smad7 is an inducible regulatory Smad protein that antagonizes TGF-₁ signal mediated via direct abrogation of R-Smad phosphorylation. The effect of overexpression of inhibitory Smad7 proteins on net collagen production was investigated using hydroxyproline assay. Our data demonstrated adenovirus-mediated Smad7 gene (Ad Smad7 at 100 MOI) transfer was associated with significant decrease of collagen synthesis in the presence and absence of TGF-₁ in primary rat cardiac myofibroblasts. In Smad7 infected cells, we also observed the ablation of TGF-₁ induced R-Smad 2 phosphorylation vs LacZ controls. Furthermore, our data revealed that overdriven Smad7 was associated with significantly increased expression of immunoreactive 65 kDa MMP-2 protein (the active form of this MMP) in culture medium of myofibroblast compared to LacZ-infected cells. Expression of the 72 kDa MMP-2 variant (i.e. inactive form of this MMP) was not altered by exogenous Smad7 transfection/overexpression. Furthermore, Smad7 overexpression was associated with a significant increase and decrease in expression of p27 and phospho-Rb protein, respectively, as well as reduced ³H-thymidine incorporation vs. Ad-LacZ infected controls. We suggest negative modulation of R-Smad phosphorylation by overdriven exogenous Smad7 adenovirus administration may contribute to the down-regulation of collagen in cardiac myofibroblasts and may suppress the proliferation of these cells. Thus treatments targeting the collagen deposition by overexpression of Smad7 may provide a new therapeutic strategy for cardiac fibrosis.