Isothermal titration calorimetry (ITC) is the only technique whereby complete set of thermodynamic profile for molecular interaction can be determined from a single experiment. Binding thermodynamic parameters (free energy, enthalpy, and entropy) provide fundamental information on the forces driving the formation of the protein-ligand complex. The connection between structural and thermodynamic features of protein-ligand binding, however, is not as simple as expected because of the multifactorial character of the enthalpy and entropy of binding. Here, we review the structural and thermodynamic studies of lipocalin-type prostaglandin D synthase (L-PGDS) as a model of broad-ligand binding properties. Our ITC and NMR analyses of interactions between L-PGDS and hydrophilic ligands, NADs, revealed that negatively charged part of ligands is important for binding to L-PGDS. Moreover, the good correlation was found between the binding free energy and hydrophobic surface area of the ligands (ASA_h), indicating that increased affinity is associated with increased ASA_h. L-PGDS has a large cavity, which contains distinct and separate hydrophilic and hydrophobic parts. Taken together, it is suggested that positively charged hydrophilic part of L-PGDS cavity is involved in ligand specificity and hydrophobic part contributes to affinity.