In order to investigate the interaction mechanism of thiocyanate with CA II, a detailed quantum chemical study using the semiempirical PM6 method was carried out. The model system used in these calculations was based on the crystal structure of human CA (PDB code – 2CBA). This system included a Zn2+ cation bonded to a HO- group and three histidine residues: His94, His96, and His119; the following residues: Glu106, Thr199, Thr200, His107, Arg246, Asn244, Gln92, and also 8 water molecules (Wat264, Wat265, Wat292, Wat318, Wat338, Wat359, Wat369, Wat389). The first step of the interaction of thiocyanate and CA occurred spontaneously with the heat of reaction ΔH = -23.7 kcal / mol. As a result of this process, the proton left the thiocyanate, there was SCN- anion formation, and proton transfer occurred through the hydrogen-bond network to the oxygen atom of residue Glu106. Further, three transition states (TS) were found. The first of these states had the activation energy Ea = 3.5 kcal / mol, and was related to the repulsion of the water molecule Wat359 from the path of the anion. The second TS occurred, when the water molecule Wat318, linked by a hydrogen bond to the hydroxyl group bonded to Zn2+, was replaced with the molecule Wat292. The activation energy for this process Ea = 4.8 kcal / mol. The third TS could be characterized by the highest activation energy, equal to 8.2 kcal / mol. In this case, the hydroxyl group bonded to Zn2+ was converted into a water molecule as a result of back transfer of a proton from the residue Thr199. After overcoming this TS, in the zinc coordination sphere there was the process of replacing the water molecule with the SCN- anion. In the equilibrium state, the Zn–N bond length was equal to 1.844 Å, and the appropriate bond order was equal to 0.639. The computational results provided a better understanding of the molecular interaction between the thiocyanate inhibitor and the CA II enzyme.