A tumor-targeting gene vector G250mAb-PEI-PEG has been prepared by modification of polyethylenimine (PEI) with polyethyleneglycol (PEG) and G250, a monoclonal antibody against the G250 antigen on tumor cell surface. The transfection efficiency was as high as 70% in G250 positive HeLa cells, whereas the transfection efficiency was relatively low (30%) in normal NIH3T3 cells. A plasmid encoding the short hairpin RNA (shRNA) specific for nucleostemin gene (NS) was efficiently transfected into the HeLa cells with this nonviral gene vector. RNA interference down-regulated the expression of NS gene in HeLa cells, inhibited cells proliferation and induced apoptosis. However, the growth and activity of the NIH3T3 cells were not affected under the same treatment. These results indicate that the reported nonviral gene vector, G250mAb-PEI-PEG, can target and efficiently deliver genes into HeLa cells, and has the potential for the cervical cancer treatment.