The purpose of this study was to investigate the potential of nuclear localization signal (NLS) “KPKKKRKV” to mediate the in vitro transfection efficiency of chitosan (CS)/DNA complexes, aiming at its use in gene therapy applications. In the preparation of CS/DNA complexes containing NLS, peptide with NLS was directly incorporated without covalent conjugation to pDNA or chitosan. The gene transfer efficiency of CS/DNA complexes with and without NLS was evaluated in the human cervical carcinoma cell line (Hela cells). The CS/DNA complex containing NLS increased transfection efficiencies in a NLS-dose dependent manner on the Hela cells, compared to the control (CS/DNA complex or NLS). The highest transfection efficiency was significantly observed in CS/DNA complex at the weight ratio of 8 with 120 μg NLS and was 74-fold higher than that in the cells transfected with CS/DNA complex. Cytotoxicity of the NLS/CS/DNA complexes increased as the amount of the peptide increased, however, over 80% average cell viability was observed for complexes at the effective concentration of the peptide for transfections. Therefore, the NLS is expected to be a potent transfection enhancing agent without a covalent conjugation to pDNA or chitosan. Our findings suggest that the high gene expression with the negligible cytotoxicity can be achieved by adding the NLS peptide to chitosan/DNA complexes at an optimal ratio.