The hierarchical self-assemblies of hydrophobically modified pullulan, such as cholesteryl-bearing pullulan (CHP) and hexadecyl group-bearing pullulan (C16P), were investigated. In a dilute aqueous solution, they formed hydrogel nanoparticles. In semidilute solution above approximately 2% (w/w), the viscosity of the CHP or C16P solution drastically increased. At higher concentration, they formed macroscopic gels: CHP gave a structure in which the nanoparticles are linked, while the C16P macrogel has mainly a fibrous network structure. In rheological measurements of CHP gel, G" exhibited a maximum at omega = 0.1 rad s(-1), while G' reached a plateau in the high-frequency regime. On the other hand, C16P gel showed G" with a peak at omega > 10 rad s(-1). The G' and G" values for CHP and C16P gels obeyed an Arrhenius plot. The activation energy of the shift factor for CHP was higher than that for C16P. Thus, we can successfully control the association of hydrophobically modified pullulans and organize them from the molecular level to the nanoscale and macroscopic region.