Obejectives To explore the effect of miR-424 on migration and invasion of non-small cell lung cancer A549 cells. Methods Expression of miR-424 in NCI-H460, NCI-H1975, NCI-H446, A549, NCI-H1299, NCI-H157 lung cancer cells and embryonic lung fibroblasts MRC-5 cells was examined by RT-PCR.miR-424 inhibitor and miR-424 NC were transfected into A549 cells with liposome Lipofectamine^TM2000. Cell viability was measured by MTT assay. Cell migration and invasion was measured by wound scratch assay and transwell migration assay,respectively.The expression of matrix metalloproteinase 2 (MMP2), MMP9, transforming growth factor-β (TGF-β1), p-Smad3 was measured by western blot. Results The expression of miR-424 in NCI-H460, NCI-H1975, NCI-H446, A549, NCI-H1299, NCI-H157 cells (1.78±0.13, 1.69±0.10, 1.89±0.18, 2.88±0.27, 2.52±0.20, 2.49±0.23) was significantly higher than that in MRC-5 cell 0.58±0.05(P< 0.01). Compared with the miR-424 NC group, the cell viability was reduced (P<0.01), cell migration and invasion ability was decreased (P< 0.01), and the expression of MMP2, MMP9, TGF-β1, p-Smad3 protein was down-regulated in miR-424 inhibitor group (P< 0.01). Conclusions miR-424 inhibitor can inhibit the migration and invasion in lung cancer A549 cells via inhibition of TGF-β1/Smad3 signal pathway.