Aim To avoid the limitation of the use of cationic polyethlenimine (PEI)-complexed plasmid DNA use for in vitro or in vivo gene delivery due to its cytotoxicity and lower efficiency in the presence of serum. Methods A polyplex with decreased positive charge on the complex surface was designed. The PEI/DNA (PD) complexes coated with an anionic biodegradable polymer, alginate were prepared and their gene delivery behavior with PD was compared. Results The alginate-coated PD polyplex, where alginate: PEI: DNA [alginate: DNA, 0. 15 (w/w); PEI: DNA, N: P = 10] showed about 10 -30 fold-increased transfection efficiency compared to corresponding non-coated complexes to C3 cells in the presence of 50% serum. The surface charge of the alginate-coated complex was approximately half of that of the alginate-lacking complex. The size of alginate-coated complex was slightly smaller than that of the corresponding complex without alginate. The former complex also showed a reduced erythrocyte aggregation activity and decreased cytotoxicities to C3 cells in comparison with PD complex. Conclusion The alginate-coated PD polyplexes as a new gene delivery system can improve transfection efficiency in high serum concentration with low cytotoxicity to C3 cells.