The electron-transfer capabilities of cytochrome b 5 reductase (Cyt b 5 R) and NADPH supply have been shown to be critical factors in microbial fatty acid synthesis. Unfortunately, Cyt b 5 R substrate specificity is limited to the coenzyme NADH. In this study, we discovered that a novel Cyt b 5 R from Mortierella alpina (MaCytb5RII) displays affinity for NADPH and NADH. The enzymatic characteristics of high-purity MaCytb5RII were determined with the K m,NADPH and K m,NADH being 0.42 and 0.07 mM, respectively. MaCytb5RII shows high specific activity at 4 °C and pH 9.0. We anchored the residues that interacted with the coenzymes using the homology models of MaCytb5Rs docking NAD(P)H and FAD. The enzyme activity analysis of the purified mutants MaCytb5RII [S230N] , MaCytb5RII [Y242F] , and MaCytb5RII [S272A] revealed that Ser230 is essential for MaCytb5RII to have dual NAD(P)H dependence, whereas Tyr242 influences MaCytb5RII's NADPH affinity and Ala272 greatly decreases MaCytb5RII's NADH affinity.