Poly(ethylene imine)-poly(l-lysine)-poly(l-glutamic acid) (PKE) polymers with various glutamic acid portions were synthesized by ring opening polymerization of l-lysine N-carboxyanhydride (NCA) and l-glutamic acid NCA from poly(ethylene imine) 1.8 kDa (PEI_1.8k) as a macroinitiator. It was found that their glutamic acid residues could buffer endosomal pH. PK₅E₉ polymer could form nanoparticles by self-assembly and nanosized polyplexes, possessing pH-responsive charge-conversion properties. PK₅E₉ or its polyplex nanoparticles showed polyhedral structures with bumpy surfaces. Its cytotoxicity was marginal at both pH 7.4 and 6.0, and its transfection efficiency was highly increased at pH 6.0. The improved transfection efficiency in acidic conditions was thought to be induced by elevated cellular uptake of the polyplexes via charge-conversion from negative to positive charges. Its transfection was also found to be mediated by endosomal escape through endosome buffering by bafilomycin A1-treated transfection. In conclusion, PK₅E₉ polymer with self-assembly and endosome buffering ability was found to possess potentials for efficient gene delivery systems in acidic conditions via charge conversion, which may be applied for tumor microenvironment-targeting.