Amphiphilic biopolymers are of interest for regenerative medicine applications due to their potential to interact with both hydrophobic and hydrophilic bioactive molecules and self-assemble into well-defined microstructures. We show that the amphiphilicity and microstructures of hydrophilic hyaluronic acid (HA) can be explicitly tuned by the stoichiometric integration of cholesterol to azide-functionalized HA via strain-promoted azide-alkyne cycloaddition (SPAAC). At low cholesterol contents, the hydrophobic interactions among the cholesterol units dynamically cross-link cholesteryl HA into physical gels showing enhanced and recoverable viscosities. By SPAAC cross-linking of remaining azides, the interdependence of physical and chemical cross-linking of cholesteryl HA is demonstrated. At higher cholesterol contents, cholesteryl HA self-assembles into multilamellar nanoparticles (NPs) composed of a core of alternately packed cholesterol-rich and HA-rich layers and a hydrated HA-rich outer layer. The amphiphilic NPs not only readily encapsulate hydrophobic compounds but also protect hydrophilic vitamin C from fast degradation in aqueous media. Rapid internalization of cholesteryl HA NPs by rat bone marrow-derived stromal cells and robust osteogenesis induced by NPs preloaded with dexamethasone and vitamin C were demonstrated. From viscoelastic physical gels, dual-cross-linked gels, to multilamellar NPs, cholesteryl HA with tailored amphiphilicity can be leveraged as versatile macromolecular building blocks for a wide range of regenerative medicine applications.