Access to the interior of the cell by destabilizing the natural barrier of the cell membrane is fundamental to drug and therapeutic delivery. Taking the unique facial amphiphilicity advantage of cholic acid to facilitate switching over impermeable cellular membranes, we report the synthesis of three different series of charge variable (anionic, cationic, and neutral) amphiphilic copolymers with various cholate contents and their membrane destabilization behavior. The synthesized cholate-containing amphiphilic copolymers self-organized into 80−100 nm sized nanoaggregates in aqueous media, while cholate moieties resided in the core of the self-assembled nanoparticles, as confirmed by 1 H NMR spectroscopy. The cholate-containing polymeric micelles proficiently destabilized the liposomes at pH 5.0 and pH 7.4. Interestingly, the cholate-appended neutral polymer exhibited up to 15fold enhancement in the membrane destabilization efficiency in comparison with respective cholate-based anionic or cationic polymers. Mechanistic studies using fluorescence, dynamic light scattering, and transmission electron microscopy established that the polymer−membrane interaction, subsequent reorganization, and insertion of cholate into the bilayer play a crucial role. Model drug (Nile red) encapsulation and release studies suggested the therapeutic potential of the cholate-conjugated polymeric nanoclusters.