Water-soluble cationic polymers, poly(histamine acrylamide)s (PHAs), with superior buffer capacity at the endosomal pH range were designed, prepared, and investigated for non-viral gene transfection. PHAs were obtained with molecular weights ranging from 9.2 to 28.7 kDa through controlled radical polymerization of histamine acrylamide (HA). Acid–base titration results displayed that all PHA polymers had a remarkably high buffer capacity of about 70% at pH 5.1–7.2. 12.7–28.7 kDa PHAs were able to effectively condense DNA into nano-sized (<220 nm) polyplexes with moderate positive surface charges (+13–+19 mV) at N/P ratios ≥10/1. CCK assays indicated that polyplexes of 12.7 and 17.5 kDa PHAs were non-toxic to COS-7 cells up to a tested N/P ratio of 20/1. Interestingly, the in vitro transfection using pCMV-Luc and pEGFP-C1 plasmid DNA as reporter genes showed that polyplexes of 12.7 kDa PHA formed at an N/P ratio of 20/1 mediated efficient transfection in COS-7 cells under 10% serum conditions, with transfection efficiencies comparable to that of 25 kDa polyethylenimine control. Their versatile design of structures, controlled synthesis, low cytotoxicity, and high transfection activity render PHA-based cationic polymers particularly interesting for the development of safe and efficient non-viral gene delivery systems. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011.