Aim To investigate pathway mechanism about transforming growth factor-β1 (TGF-β1) activated cardiomyocyte hypertrophy. Methods Fresh isolated neonatal rat cardiomyocytes induced by TGF-β1 were used to examine formation of cell pathway. p-Smad2, Smad2, Smad2/3, p15 and c-Myc proteins were measured by Western blot. Cardiomyocyte hypertrophy was measured by PI statining and RNA expressions by real-time PCR. Smad signal pathway expression and it's related protein expression were induced by TGF-β1 and siRNA interference. Results TGF-β1 could induce the expression of hypertrophic gene in cultured cardiomyocytes in vitro, and the expression of β-MHC was significantly higher than that of the control group (P＜0.01). The content of RNA in TGF-β1 group was significantly increased by PI staining (P ＜0.01). Compared with the control group, TGF-β1 significantly increased the expression of p15 and c-Myc in p-Smad2, Smad2, Smad2/3 and Smad pathway (P＜0.01). The expression of p15 of Smad2 siRNA group was less than that of TGF-β1 group (P＜0.01). Conclusion TGF-β1 may induce cardiomyocyte hypertrophy through the Smad protein pathway, and in this process the expression of p15 is increased.