Reducible disulfide-containing hyperbranched PEI-SS-HP was synthesized via click chemistry and evaluated as nonviral gene carrier. The structure of the polymers was confirmed by 1H NMR and FTIR. It was shown that the PEI-SS-HP was able to bind plasmid DNA to positively charged nanoparticles. The reduction sensitivity of the PEI-SS-HP was confirmed by gel retardation assay in the presence of DTT mimicking an intracellular reductive environment. In vitro experiments revealed that the reducible PEI-SS-HP not only had much lower cytotoxicity, but also posed superior transfection activity as compared to non-degradable 25-kDa PEI. The results indicate that a reducibly degradable PEI-SS-HP can be a promising gene vector.