To investigate the delivery of DNA into cells, lactose-poly(ethylene glycol)-grafted poly-L-lysine (Lac-PEG-PLL) polymers were synthesized as polymeric gene carriers. The new synthetic carriers, varying the substitution ratio of lactose-poly(ethylene glycol) (lactose-PEG), were characterized by NMR spectroscopy and size-exclusion chromatography. Electrophoretic mobility assay confirmed that the new gene carrier makes a complex with plasmid DNA. The attached poly(ethylene glycol) gives better solubility properties to gene/carrier complex. Transfection experiments showed that Lac-PEG-PLL efficiently delivers DNA to a hepatoma cell line in vitro; the best efficiency was achieved at a 1:3 weight ratio of DNA to carrier. As the lactose-PEG substitution content increased up to 30%, the transfection efficiency increased, which demonstrates that the lactose serves as a targeting moiety. No considerable cytotoxicity was observed due to Lac-PEG-PLL or its complex with DNA within the concentration range for this experiment. The use of chloroquine increased transfection efficiency that indicates the involvement of hydrolytic degradation of the system in lysosome. It is likely that plasmid DNA/Lac-PEG-PLL complexes enter the cells through a receptor-mediated endocytosis mechanism. These results show that Lac-PEG-PLL can form a complex with plasmid DNA and serve as an efficient gene delivery carrier with lower cytotoxicity compared to that of poly-L-lysine. Therefore, it is expected that our Lac-PEG-PLL carrier can be used as an in vivo gene delivery vector.