Retroviral vectors have recently experienced limited use in cancer gene therapy mainly due to poor transduction efficiency. To overcome this drawback, we attempted to enhance the transduction efficiency by employing different retroviral packaging cell lines and chemical additives. The retrovirus from the PG13 packaging cell line gave mostly higher or similar transduction efficiencies in a variety of human cancer cell lines compared to the retrovirus from the PA317, Bing, or FLYRD18 packaging cell line. A cationic liposome, especially Lipofectamine, significantly enhanced the transduction efficiency of a retrovirus. However, the retrovirus derived from the PG13 cell line could not infect the murine cell line efficiently even after Lipofectamine treatment. Furthermore, chloroquine did not improve the transduction efficiency regardless of the presence of chemical additives. These results, therefore, suggested that the transduction efficiency of a retrovirus in human cancer cells can certainly be improved when a proper packaging cell line is chosen. In addition, this study implied that Lipofectamine is a superb additive to enhance the transduction efficiency of a retrovirus via a specific virus envelope protein-receptor interaction for virus entry, and that receptor-mediated endocytosis does not seem to be the leading route of virus delivery to liberate a virus genome.