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Background/Aims: Hypoxia modulation of transforming growth factor (TGF)- β-induced signaling during myofibroblast transformation is dependent on the specific cell type. The purpose of this study was to explore the effects of hypoxia on myofibroblast transformation of TGF-β1-induced cardiomyocyte H9c2 cells. Methods: H9c2 cells were cultured for intermittent hypoxia treatment and TGF-β1 treatment. α-Smooth muscle actin (α-SMA) expression was examined by western blotting and immunofluorescence after treatment. To further explore the possible mechanism for this effect, the effects of hypoxia on three early TGF-β-dependent signaling pathways, i.e. the Smad2/3, RhoA and mitogen-activated protein kinase (MAPK) pathways, were screened by western blotting. Results: Intermittent hypoxia induced TGF-β1 expression, but had no effect on α-SMA expression. Exogenous TGF-β1 alone upregulated α-SMA expression in H9c2 cells in a concentration- and time-dependent manner. α-SMA expression declined with the duration of hypoxia after intermittent hypoxia and exogenous TGF-β1 co-treatment. Phospho-JNK and phospho-p38 levels were not significantly altered after TGF-β1 and hypoxia treatment. However, levels of phospho-ERK increased after TGF-β1 treatment and continued to increase after hypoxia co-treatment. The activation of phospho-Smad2/3 and phospho-RhoA induced by TGFβ1 was significantly reduced after hypoxia co-treatment. Conclusion: Hypoxia can inhibit TGF-β1-induced H9c2 myofibroblast transformation, based on inhibition of α-SMA expression by suppressing signaling downstream of TGF-β1, Smad2/3 and RhoA. It suggested that TGF-β-mediated cardiomyocyte transformation is not involved in hypoxia-mediated fibrosis.