The leech-derived anticoagulant hirudin is post-translationally sulfated on tyrosine 63, resulting in a > 10-fold increase in its affinity for thrombin. We report the structure of a biosynthetic sulfo-hirudin complexed to thrombin solved to 1.84 angstrom resolution and show that sulfation is responsible for a salt bridge and an extended hydrogen-bond network that taken together account for the increased affinity of sulfo-hirudin for thrombin. We also identify a divalent cation binding site at the interface between the two subunits of athrombin that may modulate the physiological activity of thrombin.