Abstract The host–guest interaction between Rhein (Rh) – an anthraquinonic drug characterized by low water solubility and recently considered for its potential antidiabetic and antitumoral activities other than for the well-established anti-inflammatory properties – with cyclodextrins (CDs) was investigated using phase-solubility diagrams. The typical A L phase-solubility profiles suggest the formation of the 1:1 inclusion complexes between Rh and the two CDs investigated, namely β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin and the resulting constant values of complex formation, K c, were estimated. Due to the higher K c value, complex of Rhein with 2-hydroxypropyl-β-cyclodextrin was chosen for further investigation. Characterization in solution of 2-hydroxypropyl-β-cyclodextrin/Rhein complex was achieved both by fluorescence and visible spectroscopic techniques. These results confirm the formation of inclusion complexes in solution and the 1:1 stoichiometry of the binary system. With respect to Rhein aqueous solution behavior, the inclusion complex appears to be able: (i) to enhance Rhein solubility; (ii) to control its neutral/anionic equilibrium; (iii) to affect both its electronic absorption and fluorescence spectra. Finally, the photostability of Rhein in the presence of cyclodextrins was evaluated.