In this study, gene transfection efficiencies using nano-polyplex as gene carriers were investigated in a variety of human cell lines. Gene carriers were prepared using cationic polymer methylated N-(4-pyridinylmethyl) chitosan chloride (MPyMeChC) and/or poly (ethylenimine) (PEI) to form polyplex via electrostatic interaction with 1 µg of plasmid pGL-3 basic containing CMV promoter/enhancer at different weight ratios. Polyplex formations were confirmed by gel retardation assay. The result suggested that packaging of DNA in PEI containing-polyplexes occurred without interference of DNA integrity. Both size and zeta-potential were increased when PEI was incorporated in polyplex. In vitro transfection was performed in three different cell lines which are a human cervical cancer cell line (HeLa), a human lung adenocarcinoma epithelial cell line (A549) and a human neuroblastoma cell line (SH-SY5Y). The results revealed that transfection profiles were different among the three cell lines which indicated that transfection efficiency through MPyMeChC/PEI polyplex was cell-type dependent. A synergistic effect of MPyMeChC/PEI polyplex was found in HeLa cells with the maximum transfection efficiency at a weight ratio of 1/0.5/1 providing 95% cell viability, approximately. Our study demonstrated an alternative carrier in a non-viral gene delivery system which is able to promote high gene delivery efficiency with low cytotoxicity in human cell lines.