Abstract: Objective To construct eukaryotic expression plasmids containing enhanced green fluorescent protein(EGFP) gene and serine/threonine kinase 11(STKK11) gene, and to explore its effect on migration of A549 and H460 cells. Methods Fusion plasmid of pEGFP-STK11 was constructed and identified by double restriction enzyme digestion and gene sequencing. The resultant plasmid was transfected into A549 and H460 cells respectively. The expression of EGFP and STK11 were observed by fluorescence microscopy and Western blot analysis. The cell migration ability was tested by Wound-healing assay. Results Enzyme digestion and DNA sequence analysis showed the recombinant vector of pEGFP-STK11 was constructed successfully. Fluorescence microscopy and Western blot analysis indicated STK11 gene was overexpressed in A549 and H460 cells after transfection, and reached the peak after 24 h. Wound-healing assay revealed cell migration distance of STK11 gene transfected group was significant less than that of control group (P<0.05). Conclusion STK11 gene fusion plasmid is constructed and transfected into lung cancer cell lines A549 and H460 successfully. Cells migration is inhibited to some extent after transfection.