A series of amine-functionalized diene-based polymers were synthesized via anionic and free radical polymerization techniques for evaluation of their potential as gene delivery vectors. Cytotoxicity, binding affinity, and transfection efficiency properties were examined. A systematic study of the structure-property relationships revealed that cytotoxicity decreased as the side group of the polymers changed from methyl to propyl group due to the decrease of the acidity after protonation. The binding affinity between the polymers and DNA also decreased as the size of the side group increased. Of the polymers studied, poly[2-(N,N-diethylaminomethyl)-1,3-butadiene], with lower cytotoxicity than poly( ethylene imine) and higher transfection efficiency at an N/P ratio of 2, is a promising, novel transfection vector.