Listeriolysin O (LLO), a sulfhydryl-activated pore-forming protein from Listeria monocytogenes, was tested and utilized for promoting plasmid DNA (pDNA) delivery into the cytosol of cells in culture. To render pDNA-complexing capability to LLO, the unique cysteine 484 of LLO was conjugated to polycationic peptide protamine (PN) at a 1:1 molar ratio through a reversible, endosome-labile disulfide bond. The sulfhydryl-oxidized LLO construct, LLO-s-s-PN, completely lacked its pore-forming activity, yet regained its original activity upon reduction. The enhanced cytosolic delivery using this construct therefore relies on the requisite reduction of the disulfide bond in LLO-s-s-PN by endogenous cellular reducing capacity. Condensed PN/pDNA complexes incorporating LLO-s-s-PN were tested for their enhanced gene delivery capability monitoring reporter gene expression in HEK293, RAW264.7, P388D1 cell lines and bone-marrow-derived macrophages in the presence of serum. Dramatic enhancement was observed for all tested complexes with varying weight ratios. The effect was most prominent at 0.64-0.80 (w/w) of PN/pDNA upon replacing 1-4% of PN with LLO-s-s-PN, resulting in approximately three orders of magnitude higher luciferase expression compared to PN/pDNA without apparent toxicity. These results demonstrate that incorporation of endosomolytic LLO into pDNA delivery systems in a controlled fashion is a promising approach of enhancing delivery into the cytosol of target cells in gene delivery strategies.