To clarify the effects of cavity size and substituent of cyclodextrin (CD) on the stability and the conformation of cytochrome c, thermal denaturations of cytochrome c were measured in several CD solutions at various concentrations by differential scanning calorimetry and circular dichroism. The secondary structure and the tertiary structure of cytochrome c were unfolded cooperatively in the thermal denaturation. The midpoint temperature was decreasing by addition of CD indicating that cytochrome c was destabilized by CD. The destabilization effect of CD was dependent on the interior diameter of CD, β-CD > α-CD > γ-CD, and also the substituent, mono-acetyl > methyl > 2-hydroxyl propyl. The bound number and binding constant of methyl-β-CD to the unfolded state of cytochrome c was estimated to be 5.0 ± 1.0 and 10.3 ± 2.9 M-1, respectively, indicating the stronger interaction with protein and the limited binding site on protein as compared with usual denaturants. These results indicated that CD destabilizes the folded state of cytochrome c by stabilizing the unfolded state due to inclusion of hydrophobic part of unfolded state into interior of CDs. CD will be used as a new arbitrary denaturant for protein with the combination of arbitrary cavity size and substitution of CD.