Xanthine oxidase (XO) and copper, zinc superoxide dismutase (Cu, Zn-SOD) are function-related proteins in vivo. Thermodynamics of the interaction of bovine milk XO with bovine erythrocyte Cu, Zn-SOD has been studied using isothermal titration calorimetry (ITC) and fluorescence spectroscopy. The binding of XO to Cu, Zn-SOD is driven by a large favorable enthalpy decrease with a large unfavorable entropy reduction, and shows strong entropy-enthalpy compensation and weak temperature-dependence of Gibbs free energy change. An unexpected, large positive molar heat capacity change of the binding, 3.02kJ mol−1K−1, at all temperatures examined suggests that either hydrogen bond or long-range electrostatic interaction is a major force for the binding. XO quenches the intrinsic fluorescence of Cu, Zn-SOD and causes a small red shift in the fluorescence emission maximum of the protein. A small salt concentration dependence of the binding affinity measured by fluorescence spectroscopy and a large unfavorable change in entropy for the binding measured by ITC suggest that long-range lectrostatic forces do not play an important role in the binding. These results indicate that XO binds to Cu, Zn-SOD with high affinity and that hydrogen bond is a major force for the binding.