Abstract: Objective: To construct an adenovirus vector for efficient delivery of mutant K- ras siRNA and to investigate its effect on H441 human lung cancer cells. Methods: After digestion with EcoR and Hind, the H1 promoter and K- rasV12 siRNA were cloned into the shuttle vector to construct pAdTrack- H1/siK- rasV12. Then the product vector was cotransformed with adenovirus backbone- containing plasmid pAd/PL to produce pAd/PL- H1/siK- rasV12 by homologous recombination. Recombi-nant plasmid DNA was digested with Pac and transfected into 293 cells to package adenovirus. AdH1/siK- rasV12 was transfected into H441 cells, and the inhibition of K- rasV12 gene expression was detected using western blot techniques. Results: The recombinant adenovirus AdH1/siK- rasV12 was successfully constructed. AdH1/siK- rasV12 with a titer of 4× 1011pfu/ml was obtained with CsCl gradient purification. Ninety- six hours after AdH1/siK- rasV12 transfection into H441 cells, the expression of K- rasV12 protein was significantly decreased. Conclusion: AdH1/siK- rasV12 can substantially suppress the expression of the mutant K- ras gene in H441 cells, and it provides a new method for use in gene therapy for lung cancer.