The effect of some sulfonamide ligands on the structure and function of human carbonic anhydrase II (HCA II) was investigated using different spectroscopic techniques including UV-Vis, fluorescence, circular dichroism and molecular dynamics simulation tools. Kinetic measurements were performed in 50 mM Tris-HCl, pH 7.4 at 27 °C. Kinetic data revealed that sulfonamide ligands inhibit the HCA II esterase activity in a linear competitive manner with Ki in the nanomolar range. Fluorescence measurements illustrated that ligands act as the enzyme quenchers. Stern-Volmer analysis of the quenching data at different temperatures demonstrated that the quenching of the HCA II intrinsic fluorescence occurred through static and dynamic quenching mechanisms. Analysis of the binding thermodynamic parameters showed that hydrogen bonding and hydrophobic interactions play an important role in the stabilization of enzyme-drug complex. Job plot confirmed the 1:1 stoichiometry of ligand-protein complex, and therefore, the existence of one binding site for the ligand. Molecular simulations confirmed that acetazolamide induced impressive conformational changes in two domains adjacent to the active site, including amino acids 19-25 and 61-67. RMSF studies showed sharp changes in three distinct regions near the active site including amino acids 15-25, 160-180 and 190-210 upon drug binding.