Cholesterol has been used as an effective component of therapeutic delivery systems because of its ability to cross cellular membranes. Considering this, well-defined copolymers of methacrylic acid and cholesteryl methacrylate, poly(methacrylic acid-co-cholesteryl methacrylate) P(MAA-co-CMA), were generated as potential delivery system components for pH-controlled intracellular delivery of therapeutics. Statistical copolymers with varying cholesterol contents (2, 4, and 8 mol %) were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Dynamic light scattering (DLS) analysis showed that the hydrodynamic diameters of the copolymers in aqueous solutions ranged from 5 ± 0.3 to 7 ± 0.4 nm for the copolymers having 2 and 4 mol % CMA and 8 ± 1.1 to 13 ± 1.9 nm for the copolymer having 8 mol % CMA with increasing pH (pH 4.5-7.4). Atomic force microscopy (AFM) analysis revealed that the copolymer having 8 mol % CMA formed supramolecular assemblies while the copolymers having 2 and 4 mol % CMA existed as unimers in aqueous solution. The pH-responsive behavior of the copolymers was investigated via UV-visible spectroscopy revealing phase transitions at pH 3.9 for 2 mol % CMA, pH 4.7 for 4 mol % CMA, and pH 5.4 for 8 mol % CMA. Lipid bilayers and liposomes as models for cellular membranes were generated to probe their interactions with the synthesized copolymers. The interactions were determined in a pH-dependent manner (at pH 5.0 and 7.4) using surface plasmon resonance (SPR) spectroscopy and liposome leakage assay. Both the SPR analyses and liposome leakage assays indicated that the copolymer containing 2 mol % CMA displayed the greatest polymer-lipid interactions at pH 5.0, presenting the highest binding ability to the lipid bilayer surfaces, and also demonstrating the highest membrane destabilization activity. CellTiter-Blue assay showed that the copolymers did not affect the cell viability up to 30 μM over a period of 72 h.