A polyethylenimine-poly(hydroxyethyl glutamine) copolymer (PEI-PHEG) was designed and synthesized as a gene delivery system. The molecular structure of PEI-PHEG was characterized using nuclear magnetic resonance. Moreover, PEI-PHEG/pDNA complexes were fabricated and characterized by gel retardation assay, particle size analysis, and zeta potential analysis. The transfection efficiency and cytotoxicity of PEI-PHEG were evaluated using human cervical carcinoma (HeLa), human embryonic kidney (HEK293), and murine colorectal adenocarcinoma (CT26) cells in vitro. The results show that PEI-PHEG could effectively form positively charged nanosized particles with pDNA; the particle size was in a range of 130.2 to 173.0 nm and the zeta potential was in a range of 27.6 to 41.0 mV. PEI-PHEG exhibited much lower cytotoxicity and higher gene transfection efficiency than PEI-25K with different cell lines in vitro. An animal test was also conducted on a Lewis Lung Carcinoma tumor model in C57/BL6 mice by using subcutaneous intratumoral administration. The results show that in vivo transfection efficiency of PEI-PHEG was improved greatly compared with that of commercial PEI-25K. These results demonstrate that PEI-PHEG can be a potential nonviral vector for gene delivery systems both in vitro and in vivo. (C) 2011 Wiley Periodicals, Inc. J Appl Polym Sci 123: 2257-2265, 2012