AbstractMetabolic engineering of Saccharomyces cerevisiae for xylose fermentation has often relied on insertion of a heterologous pathway consisting of nicotinamide adenine dinucleotide (phosphate) NAD(P)H‐dependent xylose reductase (XR) and NAD+‐dependent xylitol dehydrogenase (XDH). Low ethanol yield, formation of xylitol and other fermentation by‐products are seen for many of the S. cerevisiae strains constructed in this way. This has been ascribed to incomplete coenzyme recycling in the steps catalyzed by XR and XDH. Despite various protein‐engineering efforts to alter the coenzyme specificity of XR and XDH individually, a pair of enzymes displaying matched utilization of NAD(H) and NADP(H) was not previously reported. We have introduced multiple site‐directed mutations in the coenzyme‐binding pocket of Galactocandida mastotermitis XDH to enable activity with NADP+, which is lacking in the wild‐type enzyme. We describe four enzyme variants showing activity for xylitol oxidation by NADP+ and NAD+. One of the XDH variants utilized NADP+ about 4 times more efficiently than NAD+. This is close to the preference for NADPH compared with NADH in mutants of Candida tenuis XR. Compared to an S. cerevisiae‐reference strain expressing the genes for the wild‐type enzymes, the strains comprising the gene encoding the mutated XDH in combination a matched XR mutant gene showed up to 50% decreased glycerol yield without increase in ethanol during xylose fermentation.