Objective: To explore the enhanced cell killing effect of HSV tk using VP22 intercellular traffciking. Methods: The chimeric genes were constructed by fusing a marker gene for the green fluorescent protein (GFP) or a prodrug enzyme gene for the Herpes simplex virus thymidine kinase (HSV tk) with that of VP22. After being sequenced, the fusion genes were transferred into 293T or COS7 cells. The transfection efficiency and intercellular trafficking were certified using Western blot and immunofluorescence.The cell proliferation was detected through MTT method in the different concentration of GCV and under indicated between transfected cells and untransfected cells. The supernatant of transfected cells was used to culture the untransfected cells to test whether the bystander effect could transferred by media. Results: The gene insertion was proved correct using PCR and DNA sequencing. When the fusion genes were transferred into 293T or COS7 cells at transfection efficiency of 25%～30%, fusion proteins were expressed and efficient intercellular trafficking was demonstrated.The VP22 HSV tk, as a prodrug enzyme fused with VP22, showed an amplified cell killing effect in the presence of GCV as low as 0.1 ?g/ml. Further quantification of the bystander effect showed that cell killing increased with higher proportion of VP22 HSV tk expressing cells. The bystander effect could not be transferred through media. Conclusion: These results clearly indicate that VP22 enhanced intercellular trafficking promotes tumor cell killing effect of HSV tk/GCV.