Hydrogenases (H 2 ase) catalyze the oxidation of dihydrogen and the reduction of protons with remarkable efficiency, thereby attracting considerable attention in the energy field due to their biotechnological potential. For this simple reaction, [NiFe] H 2 ase has developed a sophisticated but intricate mechanism with the heterolytic cleavage of dihydrogen, where its NiÀ Fe active site exhibits various redox states. Recently, new spectroscopic and crystal structure studies of [NiFe] H 2 ases have been reported, providing significant insights into the catalytic reaction mechanism, hydrophobic gas-access tunnel, protontransfer pathway, and electron-transfer pathway of [NiFe] H 2 ases. In addition, [NiFe] H 2 ases have been shown to play an important role in biofuel cell and solar dihydrogen production. This concept provides an overview of the biocatalytic reaction mechanism and biochemical application of [NiFe] H 2 ases based on the new findings.