The significance of halogen bonding in protein-ligand interactions has been recognized recently. We present here the first comprehensive thermodynamic and structural characterization of halogen bonding in PDE5-inhibitor interactions. ITC studies reveal that binding strength of the halogen bonding between chlorine, bromine, and iodine of inhibitor and the protein is -1.57, -3.09, and -5.59 kJ/mol, respectively. The halogens interact with the designed residue Y612 and an unexpected buried water molecule.