Due to their unique structure and properties, water-soluble fullerene derivatives are of great interest for various biomedical purposes. In this work, solution behavior, encapsulation and release properties, biocompatibility, and cellular uptake pathways of fullerene-polyglycerol amphiphiles (FPAs) with defined structures are investigated. The number of polyglycerol branches attached to the surface of fullerene affects the physicochemical properties of FPAs dramatically but not their cellular uptake. Release of encapsulated hydrophobic dyes from FPAs strongly depends on the number of their branches. Conjugation of a pH-sensitive dye to the FPAs as a probe showed that their self-assemblies are taken up through endocytotic pathways. It was observed that FPAs are able to transfer small molecules into cells both above and below their critical aggregation concentration. Taking advantage of the water solubility, biocompatibility, and transfer-ability of FPAs, they might find use as unimolecular carriers for future biomedical applications.