The use of polyethylene glycol (PEG)-modified lipids (PEG-lipids) as a component of cationic liposomes impairs the cytoplasmic delivery of the encapsulated cargos by reducing endosomal escape. While this results in a loss of gene expression of encapsulated plasmid DNA, PEG-modification is useful in that it permits the formation of small, stabilized particles. In the present study, the dilemma associated with the use of PEG was overcome by modifying liposomes with stearylated INF7 (STR-INF7), a membrane fusion-independent destabilizer of endosomes, and substituting hydrophobic lipid-anchors in the PEG-lipid. The cationic liposomes modified with a series of PEG-lipids showed a drastically impaired transgene expression. However, the incorporation of STR-INF7 recovered the gene expression, and this was found to be mainly dependent on the type of PEG lipid-anchor used. Of note, the fold increase in transfection activity was highest in cholesterol-anchored PEG (>100-fold), whose enhanced endosomal escape was followed by imaging techniques. These data suggest that the structure of lipid-anchors in PEG affects the action of the peptides for inducing of endosomal escape.