Polyrotaxanes are supramolecular assemblies consisting of cyclic molecules (e.g., α-cyclodextrins) and linear polymer chains (e.g., poly[ethylene glycol]), in which cyclic molecules can move along the polymer chain. Here, we examined the effect of functional groups introduced into the α-cyclodextrins of polyrotaxane on cell responses such as adhesion, proliferation, and differentiation. Polyrotaxane-based triblock copolymers modified with methyl (CH3, hydrophobic, and nonionic), hydroxy (OH, hydrophilic and nonionic), amino (NH2, cationic), and sulfo (SO3H, anionic) groups were coated on the surface of the culture plate to fabricate polyrotaxane surfaces with different surface chemistries. The chemical compositions of each surface were determined via time-of-flight secondary ion mass spectrometry and X-ray photoelectron spectroscopy. The contact angle hysteresis reflecting the molecular mobility and zeta potential of each polyrotaxane surface changed depending on the functional groups. When osteoblast and adipocyte differentiation was induced in human mesenchymal stem cells cultured on each polyrotaxane surface, the cells adhered to the SO3H-modified polyrotaxane surfaces exhibited osteoblast differentiation, whereas the cells adhered to the OH-, NH2-, and SO3H-modified polyrotaxane surfaces preferentially underwent adipocyte differentiation compared with those on the unmodified and CH3-modified polyrotaxane surfaces. Interestingly, the SO3H-modified polyrotaxane surfaces promoted both osteoblast and adipocyte differentiation. High molecular mobility and negative charge on the SO3H-modified polyrotaxane surfaces are expected to contribute to the facilitation of both osteoblast and adipocyte differentiation.