Objective To investigate the effect of Smad3 on cell migration of A549 and HeLa cells. Methods Primers for pCMV-Myc-Smad3 plasmid construction and siRNA targeting Smad3 were designed and synthesized. pCMV-Myc-Smad3 plasmid was constructed with molecular cloning techniques. Overexpression of Smad3 with Myc-tag or silencing of endogenous Smad3, and then scratch assay was used to detect the migration ability of A549 and HeLa cells in vitro. Results pCMV-Myc-Smad3 plasmid was successfully constructed. Overexpression of Smad3 significantly up-regulated the migration rate of A549 and HeLa cells. Conversely, in the same cells, silencing of endogenous Smad3 or treatment with Smad3 inhibitor, SIS3, down-regulated the migration rate. Conclusions Smad3 promotes cell migration of A549 and HeLa cells.