Transforming growth factor (TGF) ₁ is one of the most pleiotropic and multifunctional peptides known. While the cardioprotective effect of TGF₁ during ischemia is well-known, the specific role of TGF₁ in altering the cardiac remodeling process remains unclear. This study was designed to examine the regulation of hypoxia-reoxygenation-mediated collagen type I expression and activity of matrix metalloproteinases (MMPs) by over-expression of TGF₁ in cultured HL-1 mouse cardiomyocytes. TGF₁ was over-expressed in cardiomyocytes by transfection with AAV/TGF₁ Latent or with AAV/TGF₁ CT (active TGF₁). Twenty four hours of hypoxia followed by 3 hours of reoxygenation (H-R) markedly enhanced (pro-)collagen type I expression and activity of MMPs concomitant with an increase in reactive oxygen species (ROS) release and LOX-1 expression. Over-expression of TGF ₁ reduced these alterations induced by H-R. TGF₁ over-expression also blocked H-R mediated p38 and p44/42 MAPK activation. Transfection with AAV/TGF₁ ACT was superior to that with AAV/TGF₁ Latent. These data for the first time demonstrate that H-R induces signals for cardiac remodeling in cardiomyocytes and TGF₁ can modulate, possibly via anti-oxidant mechanism, these signals. These findings contribute to further understanding of the role of TGF₁ in the cardiac remodeling process.