Atomic force microscopy (AFM) was used to monitor the reductive disassembly of DNA/polypeptide polyplexes in real time. Reducible cationic polypeptides with disulfide bonds in the backbone were used to condense plasmid DNA into nanosized polyplexes. Polypeptides based on a histidine-rich peptide (HRP, amino acid sequence CKHHHKHHHKC) and a nuclear localization sequence peptide ((NLS, amino acid sequence CGAGPKKKRKVC) were synthesized. The polypeptides are capable of condensing the DNA into positively charged nanoparticles. The positively charged nanoparticles adsorb at the aqueous solution and mica interface. The polyplexes were observed to decondense and desorb in response to a reducing environment. The DNA release is triggered by the depolymerization of the polypeptides at the disulfide bond. AFM proves to be effective in revealing the molecular pathways of DNA release from the polyplexes in response to the reducing stimulus. The results contribute to the design of more efficient gene delivery vectors.