Metal ions play essential roles in protein structures, function, and stability. Practically, over one third of proteins from Protein Data Bank (PDB) contain metal ions, and approximately 40 % of enzymes for which three-dimensional structures are known required metal ions for their activities. Metal ions play roles as cofactors for enzymes and regulate functions of enzymes by binding to the enzymes, except as cofactors. Therefore, knowledge of metal-enzyme interactions is essential for elucidating the function and reaction mechanism of enzymes. Metal-binding has been identified by such experimental approaches as isothermal titration calorimetry (ITC), circular dichroism, differential scanning calorimetry, absorbance spectroscopy, and X-ray. Among them, ITC is particularly a fundamentally essential technique to study metal-enzyme interactions, because it can reliably assess the thermodynamic underpinnings of binding events, and it is not restricted by the photophysical properties of a metal. In this review, we highlight the analysis of enzyme-metal interactions based on ITC about two enzymes, carbonic anhydrase II (CAII) and cutinase-like enzyme (Cut190), in which metal ions are playing different roles as a cofactor and activating/stabilizing factors, respectively. We evaluate and elucidate analytical results and propose the additional plans, which complement ITC in case metal binding is too weak for reliable assessment.