The aim of this research is to develop a novel branched polyethylenimine (PEI)-like polycation as a potential gene carrier with high gene transfection efficiency and low toxicity. In particular, alpha,beta-poly(l-aspartate-graft-PEI) (Asp-g-PEI), a pseudo-branched PEI, was synthesized by the ring-opening reaction of poly(l-succinimide) (PSI) with low molecular weight branched PEI (LMW PEI, MW=600 and 1200). Good plasmid condensation and protection ability of Asp-g-PEI were confirmed by agarose gel electrophoresis assay. Asp-g-PEI/DNA complexes showed high positive zeta potential, narrow size distribution, good dispersity and a compact spherical shape with size below 250nm when the N/P ratio was above 5, suggesting that they can be endocytosed. Cytotoxicity of Asp-g-PEI/DNA complexes was rather lower than that of PEI25K/DNA complexes, especially at high N/P ratio. The most efficient gene transfection of Asp-g-PEI/DNA complexes was similar or a little higher than that of PEI25K in 293T, HeLa and HepG2 cell lines, while almost 4 and 6 times higher than that of parent PEI1200 and PEI600, respectively, in HeLa cell line; as the molecular weight of parent PEI in Asp-g-PEI was increased from 600 to 1200, the transfection efficiency showed a tendency to decrease. The mechanism of Asp-g-PEI-mediated gene transfection was attributed to the "proton sponge effect" due to PEI in the copolymer.