Development of efficient xylose-fermenting Saccharomyces cerevisiae strain has involved a large number of trials that focus on improving ethanol production from glucose and xylose present in lignocellulosic hydrolysates. In this study, a recombinant S. cerevisiae strain (SK-NY) overexpressing GRE3-encoded NADPH-dependent aldose reductase and xylulokinase with a mutated strictly NADP(+)-dependent Pichia stipitis xylitol dehydrogenase was constructed and its fermentation efficiency was compared with that of an isogenic constructed reference strain expressing P. stipitis xylose reductase instead of the GRE3 gene. Strain SK-NY efficiently fermented xylose and glucose mixture and the ethanol production by SK-NY was 21.4% higher than that of the reference strain. Interestingly, the yield of ethanol production by SK-NY strain increased from 0.395 g ethanol/g sugar to 0.435 g ethanol/g sugar after glucose depletion. Furthermore, xylitol accumulation by SK-NY strain (0.6% of total sugar) was considerably lower than that of the reference strain (4.8% of total sugar). These improvements may be influenced by the effective regeneration of NADPH/NADP(+) cofactors by GRE3 gene and the mutated strictly NADP(+)-dependent P. stipitis xylitol dehydrogenase. (C) 2014 Elsevier Ltd. All rights reserved.