Polyaspartamides containing pendant imidazolyl propyl groups and amino ethyl groups were synthesized via ring-opening of polysuccinimide (PSI) reacting with N-Boc protected ethylenediamine and 1-3-aminopropyl) imidazole. Polyaspartamides (P1 similar to P5) with different grafting ratios were characterized by FTIR and (1)H-NMR spectra, which were well in accordance with the expected chemical structures. The cytotoxicity of polymers was evaluated by MTT assay. Compared to the commercial branched polyethyleneimine (PEI) (25 kDa) with an IC50 of 15 mu g/mL, P1 similar to P5 showed no significant cytotoxicity against both 293T cells and COS-7 cells. More than 90% cells still retained their metabolic ability even at the polymer concentration up to 0.8 mg/mL. The introduction of imidazolyl group into polymer side chains make them possess high buffer capacity between pH 5 and pH 7, which was 2 similar to 5 times higher than that of PEI. Gel electrophoresis assay and dynamic light scattering measurements were performed to characterize the polycation/DNA complexes. At an N/P ratio of above 5, all of the five polyaspartamide-based polycations can condense plasmid DNA to form nanosized (100 nm) polyelectrolyte complexes with positive surface charge (+20 mV). The transfection assay demonstrated that the transfection efficiencies of foreign DNA in 293T cells and COS-7 cells increased with increasing the contents of imidazolyl graft of polyaspartamides. P4 with a grafting ratio of 1/3.7 (amino ethylene group to imidazolyl propyl group) showed the highest ability to deliver pGL-3 and pEGFP-C1 into both 293T cells and COS-7 cells. At the optimal N/P ratios, the gene expression efficiencies mediated by P4 were comparable to or even 2 similar to 3 times higher than that of PEI. The results suggest that imidazole-containing polyaspartamides are a very promising class of novel polycations for highly efficient and less toxic gene delivery. The structure-gene delivery ability relationship of imidazole-grafted polyaspartamides will provide valuable insight into the development of novel nonviral gene carrier.