A series of reducible hyperbranched poly(amido amine)s (RHB) with high transfection efficiency were designed and synthesized as nonviral gene delivery vectors. The polycations were synthesized by Michael addition copolymerization of N,N-dimethylaminodipropylenetriamine (DMDPTA) and two bisacrylamide monomers N,N'-hexamethylene bisacrylamide (HMBA) and N,N'-cystamine bisacrylamide (CBA). The density of disulfide linkages in the synthesized hyperbranched polymers was tuned by varying the feed molar ratio of the bisacrylamide monomers. The results demonstrate that disulfide content in RHB controls the molecular weight of the polycation degradation products, ease of polyplex disassembly, polycation cytotoxicity, and polyplex transfection activity. Due to their lower cytotoxicity, polyplexes based on reducible polycations could be used safely in a wider range of DNA doses than nonreducible controls. As a result, significantly increased transfection activity is achieved with optimized formulations of reducible polyplexes compared with nonreducible controls.