To improve gene transfection efficiency in vitro and in vivo, we first grafted polyglycine (PGly) onto branched polyethyleneimine (PEI) (molecular weight M-W 25 kDa) to obtain a novel gene vector PGly-PEI (PPG), before complexing it with poly(ethylene glycol)-poly-l-glutamate (PEG-PLG) for adjusting the charge density of PEI. We named the resulting nano-polyplex PG/PPG. The PPGs demonstrated improved gene transfection efficiency after grafting with polyglycine, and the efficiency could be further increased with the introduction of PEG-PLG. The PG/PPG exhibited not only excellent DNA and siRNA transfection capacity in various cell lines including HeLa, CHO, HepG2, MCF7 and Huh7, but also reduced cytotoxicity compared with PEI. Besides, PG/PPG induced a higher degree of cell apoptosis when mixed genes of pKH3-rev-casp-3 plasmid and siBcl2 were used rather than either of them. Furthermore, PG/PPG was highly resistant to serum, which led to enhanced in vivo red fluorescent protein (RFP) expression in HeLa tumors. These results suggested that PG/PPG is a promising gene carrier for highly efficient gene delivery.