Objective: To investigate the effect of Smad4 specific small interference RNA (siRNA) on the invasive ability and MMP-9 expression of the breast cancer MDA-MB-231 cells. Methods: After Smad4 siRNA construction, the breast cancer cells were divided into control group, Smad4 siRNA transfection group, TGF-β treatment group and Smad4 siRNA transfection plus TGF-β treatment group. The expression of Smad4 protein in the breast cancer cells transfected with Smad4 siRNA was tested by Western blot analysis, and the activity of 4xSBE (Smad4 binding element) of the cells after TGF-β treatment was detected by luciferase reporter gene assay. Subsequently, the cell invasive ability and MMP-9 expression of the cells of each group were determined by using Transwell chamber system and Western blot analysis, respectively. Results: The protein expression of Smad4 and the TGF-β-induced elevation of 4xSBE activity of the breast cancer cells were significantly inhibited by Smad4 siRNA transfection (both P<0.05). The invasive ability and MPP-9 expression level of the breast cells stimulated by TGF-β were significantly increased compared with the cells of control group (both P<0.05), but these effects of TGF-β were significantly inhibited in the cells previously transfected with Smad4 siRNA (both P<0.05). The Smad4 siRNA transfection alone had no obvious effects on the invasive ability and MMP-9 expression of the cells. Conclusion: Smad4 siRNA can effectively decrease the invasive ability of MDA-MB-231 cells promoted by TGF-β1, and this effect may be associated with its inhibition of MMP-9 expression.