NAD(P)H regeneration is important for biocatalytic reactions that require these costly cofactors. A mutant phosphite dehydrogenase (PTDH-E175A⁄A176R) that utilizes both NAD and NADP efficiently is a very promising system for NAD(P)H regeneration. In this work, both the kin-etic mechanism and practical application of PTDH-E175A⁄A176R were investigated for better understanding of the enzyme and to provide a basis for future optimization.