Sudlow Site I of human serum albumin (HSA) is located in subdomain IIA of the protein and serves as a binding cavity for a variety of ligands. In this study, the binding of warfarin (W) is examined using computational techniques and isothermal titration calorimetry (ITC). The structure of the docked warfarin anion (W⁻) to Site I is similar to that revealed by X-ray crystallography, with a calculated binding constant of 5.8 × 10⁵ M⁻¹. ITC experiments (pH 7.13 and I = 0.1) carried out in three different buffers (MOPs, phosphate and Tris) reveal binding of W⁻ is accompanied by uptake of 0.30 ± 0.02 protons from the solvent. This measurement suggests that the binding of W⁻ is stabilized by an ion-pair interaction between protonated H242 and the phenoxide group of W⁻.