pH-responsive liposomes were prepared by modifying the liposome with acid-cleaving amphiphiles. Palmitic ketohydrazone (P-KH) or stearic ketohydrazone (S-KH), composed of hydrophilic sugar headgroup and hydrophobic acyl chain, was used as a modifier of the DMPC liposome. Because the ketohydrazone group of P-KH or S-KH was cleaved at low pH conditions (<pH 5.0), the delivery of the P-KH modified liposomes was observed probably via an endocytic pathway. The membrane properties of these liposomes were characterized, focusing on the variation of both polarity (measured by Laurdan) and membrane fluidity (measured by DPH) at low pH condition. The interface of the P-KH modified liposome at acidic pH was found to become more hydrophobic and less fluidic as compared with that at neutral pH; that is, P-KH modified liposome became more rigid structure. Therefore, it seems that the P-KH modified liposome could protect encapsulated drugs from the enzymes in the lysosome. This study shows the novel approach about design of pH-responsive liposomes based on the membrane properties.