Multicellular tumor spheroids (MCTSs) are attractive for drug screening before animal tests because they emulate an in vivo microenvironment. The permeability of the MCTSs and tumor tissues towards the candidate drugs is not sufficient even though the drugs can penetrate monolayer cultured cells; therefore, nanocarriers are required to enhance permeability and deliver drugs. In this study, we prepared zwitterionic polymers of sulfobetaine methacrylates and (meth)acrylamides with or without hydroxy groups between the zwitterions to serve as highly permeable nanocarriers. In the sulfobetaine polymers, poly(2-hydroxy-3-((3-methacrylamidopropyl)dimethylammonio)propane-1-sulfonate), P(OH-MAAmSB), the hydroxy group containing methacrylamide polymer exhibited little cytotoxicity and membrane translocation ability against monolayer cultured cells. Moreover, the excellent permeability of the hepatocyte MCTS enabled P(OH-MAAmSB) to permeate it and reach the center region (∼325 μm in diameter) at approximately 150 s, although poly(trimethyl-2-methacroyloxyethylammonium), a cationic polymer, penetrated just 1 to 2 layers from the periphery. The superior permeability of P(OH-MAAmSB) might be due to its good solubility and side chain conformation. P(OH-MAAmSB) is a promising nanocarrier with membrane translocation and permeability.