© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Endocytic pathways are practical routes for the intracellular delivery of biomacromolecules. Along with this, effective strategies for endosomal cargo release into cytosol are desired to achieve successful delivery. Focused on compositional differences between the cell and endosomal membranes and the pH decrease within endosomes, we designed the lipid-sensitive and pH-responsive endosome lytic peptide HAad. This peptide contains aminoadipic acid (Aad) residues, which serve as a safety-catch for preferential permeabilization of endosomal membranes over cell membranes, and His-to-Ala substitutions to enhance the endosomolytic activity. The ability of HAad to destabilize endosomal membranes was also supported by the model studies using large unilamellar vesicles (LUVs) and by increased intracellular delivery of biomacromolecules (including antibodies) into live cells. Cerebral ventricle injection of Cre recombinase with HAad led to Cre/loxP recombination in a mouse model, showing potential applicability of HAad in vivo.