The relatively low efficiency of transgene expression is a major drawback of non-viral gene delivery systems despite it being a safer alternative for gene therapy. Modifications of non-viral carriers with peptides have been utilized to improve their gene transfer efficiency. In this study, we described a new combined carrier, which was comprised of a cationic polymer, polyethylenimine (PEI), and a nuclear protein, high mobility group box 1 (HMGB1) containing nuclear localization sequences (NLS). The HMGB1/branched or linear PEI (b-PEI or l-PEI) combined carriers have been investigated for their cytotoxicity and ability to condense and deliver plasmid DNA (pDNA) to mammalian cells in vitro. Both HMGB1 and PEI formed complexes with pDNA are revealed by a gel-retardation assay. Compared with pDNA/l-PEI complexes, a reduction in particle size was observed for pDNA/HMGB1/l-PEI ternary complexes. In MTT assay the results of cell viability suggested lower cytotoxicity for HMGB1/PEI combined carriers. Transfection efficiencies of pDNA/HMGB1/PEI ternary complexes were evaluated by green fluorescent protein expression level measured by fluorescence spectroscopy and flow cytometry. Transfection efficiencies for pDNA/HMGB1/l-PEI ternary complexes and pDNA/HMGB1/b-PEI ternary complexes showed more than 2.9-fold and 4.0-fold greater than that for pDNA/l-PEI complexes and pDNA/b-PEI complexes, respectively. Thus, our results showed that HMGB1/PEI combined carriers may be a versatile non-viral carrier for pDNA with high transfection efficiency.