The catalytic mechanism of an Fe-containing nitrile hydratase (NHase) subsequent to the formation of a cyclic intermediate was investigated using a hybrid quantum mechanics/molecular mechanics (QM/MM) method. We identified the following mechanism: (i) proton transfer from βTyr72 to the substrate via αSer113, and cleavage of the S-O bond of αCys114-SO(-) and formation of a disulfide bond between αCys109 and αCys114; (ii) direct attack of a water molecule on the sulfur atom of αCys114, which resulted in the generation of both an imidic acid and a renewed sulfenic cysteine; and (iii) isomerization of the imidic acid to the amide. In addition, to clarify the role of βArg56K, which is one of the essential amino residues in the enzyme, we analyzed a βR56K mutant in which βArg56 was replaced by Lys. The results suggest that βArg56 is necessary for the formation of disulfide intermediate by stabilizing the cleavage of the S-O bond via a hydrogen bond with the oxygen atom of αCys114-SO(-).