A novel non-viral gene delivery system comprised of a DNA/PEI/Alginate (DPA) polyplex was prepared and assessed in vitro and in vivo. Coating the positively charged DNA/PEI (DP) complex with a polyanion resulted in a high level of in vitro reporter gene transfection in the presence of 50 vol% serum due to the minimized cytotoxicity of PEl and the reduced nonspecific interactions with serum components. Among the tested anionic polymers, which included sodium alginate, poly(methacrylic acid) and poly(acrylic acid), the sodium alginate showed the highest gene transfection efficiency. The DPA polyplex also showed a reduced level of erythrocyte aggregation in target cells when compared with the DP complex. According to in vivo studies in which reporter genes encoding green fluorescent protein (GFP) and luciferase were used, injection of the DPA polyplex into tumor cells in six week old female C57/BL6 mice resulted in a much higher level of GFP expression and approximately 7 fold higher luciferase expression than treatment with the DP complex. Taken together, these results demonstrated that the anionic alginate coating of the DP complex contributed to efficient gene delivery in vitro and in vivo.