Endosomal escape after endocytosis is a critical step for protein-based agents to exhibit their effects in the cytosol of cells. However, antibodies internalized into cells by endocytosis cannot reach the cytosol due to their inability to escape from endosomes. Here, we report a unique endosomal escape mechanism of the IgG-format TMab4 antibody, which can reach the cytosol of living cells after internalization. Dissociation of TMab4 from its cell surface receptor heparan sulfate proteoglycan by activated heparanase in acidified early endosomes and then local structural changes of the endosomal escape motif of TMab4 in response to the acidified endosomal pH were critical for the formation of membrane pores through which TMab4 escaped into the cytosol. Identification of structural determinants of endosomal escape led us to generate a TMab4 variant with ~3-fold improved endosomal escape efficiency. Our finding of the endosomal escape mechanism of the cytosol-penetrating antibody and its improvement will establish a platform technology that enables a full-length IgG antibody to directly target cytosolic proteins. TMab4 cytotransmab undergoes conformational changes in response to the acidified pH of early endosomes for the membrane pore formation through which TMab4 escapes into the cytosol Display omitted