A series of novel water-soluble, bioreducible poly(amido amine)s containing disulfide linkages in their amino units (SS-PAAs) was synthesized by Michael addition polymerization of N,N′-dimethylcystamine (DMC) with various bisacrylamides. The synthetic route allows large structural variation in the bisacrylamide segments and is complementary to the earlier developed route to SS-PAAs in which the disulfide bond is incorporated in cystamine bisacrylamide units. The physicochemical and biomedical properties of the novel DMC-based polymers were evaluated for their application as non-viral gene delivery vectors and compared with analogs lacking the disulfide moieties. DMC-based SS-PAAs show high buffer capacities in the pH range pH 5.1–7.4, a property that may favorably contribute to the endosomal escape of the polyplexes. The polymers are capable to condense DNA into nanoscaled (< 250 nm) and positively charged (>+ 20 mV) polyplexes which are relatively stable in medium mimicking physiological conditions but rapidly disintegrate in the presence of 2.5 mM DTT, mimicking the intracellular reductive environment. Polyplexes from DMC-based SS-PAAs are capable to transfect COS-7 cells in vitro with transfection efficiencies up to 4 times higher than those of pDMAEMA and PEI, with no or only very low cytotoxicity at the polymer/DNA ratios where the highest transfection is observed. The results show that DMC-based SS-PAAs have very promising properties for the development of potent and non-toxic polymeric gene carriers.