Molecular dynamics (MD) simulations and free energy calculations were integrated to investigate substrate-enzyme dynamic interactions during the unbinding of phenylsulfonamide from carbonic anhydrase II (CA II). The potential of mean force (PMF) along the unbinding pathway shows that a special ligand-binding state exists, and the electrostatic interaction dominates the ligand?s binding with CA II. The analysis of trajectories reveals that, apart from the zinc ion, the key residues in the unbinding pathway, Leu198, Thr199, and Thr200, prevent the substrate?s unbinding from the enzyme by hydrogen bonding with the sulfanilamide group of the substrate. The present results are of direct significance for the in-depth understanding of the sulfonamide-CA II binding process and related drug design.