Aim To investigate the effects of Smad3 on the growth of C2C12 cells induced by transforming growth factor-β1 (TGF-β1). Methods C2C12 cells were transfected with siRNA-Smad3 in vitro, and the cell proliferations were examined with MTT methods. Results After the C2C12 cells interfered with different concentrations of TGF-β1 (0, 1, 5, 10 μg/L) for 24 h, the MTT (OD) values were 0.096±0.015, 0.177±0.014, 0.240±0.028, 0.312±0.012 respectively (P<0.01). While the C2C12 cells were transfected with 200 pmol/L siRNA-Smad3 for 24 h and then treated with 5 μg/L of TGF-β1, the MTT(OD) values in experimental group (0.063±0.011) were decreased compared with internal control group (0.137±0.016, P<0.01). Conclusion The TGF-β1 could promote C2C12 cells proliferation by the Smad3 pathway and showed dose dependent manner, siRNA-Smad3 could block the signal transduction and decreased C2C12 cell proliferation effectively.