To clarify the effect of cyclodextrin (CD) on the stability of cytochrome c, the thermal denaturation of cytochrome c was measured by differential scanning calorimetry in aqueous solutions of β-CD modified with three substituents: methyl, acetyl, and 2-hydroxylpropyl groups. The midpoint temperature of thermal denaturation decreased with the addition of modified β-CDs, indicating that cytochrome c was destabilized. The destabilization effect of CD depended on the substituent and increased in the order of acetyl>methyl>2-hydroxypropyl. The estimated binding number and binding constant of the modified β-CDs for cytochrome c are 5.0 ± 1.0 and 10.3 ± 2.9 M−1 for methyl-β-CD, 13.8 ± 3.6 and 4.7 ± 1.6 M−1 for acetyl-β-CD, and 2.8 ± 0.9 and 7.0 ± 3.0 M−1 for 2-hydroxypropyl-β-CD. The destabilization effect of acetyl-β-CD is the highest because many CD molecules interact with proteins by the inclusion effect of CD and the inhibition effect of the acetyl group on the hydrogen bond in the secondary structure. In contrast, the stabilization effect of 2-hydroxypropyl-β-CD is the smallest because the steric exclusion of the 2-hydroxypropyl group inhibits the binding of CD to cytochrome c as compared with the smaller structure of the methyl group. Dependency of the destabilization effect on the molar ratio of CD to cytochrome c suggests that the destabilization effect of CD is caused not only by the “direct” interaction of CD with proteins but also by the “indirect” interaction of CD which promotes the solvation of hydrophobic groups by altering the water structure as observed in urea.