Carboxymethyl poly( l-histidine) (CM-PLH) as a new pH-sensitive polypeptide has enhanced polyplex gene delivery. Agarose gel retardation assay and zeta potential measurement proved that the anionic CM-PLH at physiological pH coated the PEI/DNA binary complexes. The resulting CM-PLH/PEI/DNA ternary complexes showed the gene expression value 300 times higher than that of the PEI/DNA binary complexes. These results suggest that the synergistic effect of the pH-sensitive imidazole groups at endosomal pH and the anionic carboxymethyl groups at physiological pH in the CM-PLH enhanced polyplex gene delivery.