The co-production of xylitol and ethanol from agricultural straw has more economic advantages than the production of ethanol only. Saccharomyces cerevisiae, the most widely used ethanol-producing yeast, can be genetically engineered to ferment xylose to xylitol. In the present study, the effects of xylose-specificity, cofactor preference, and the gene copy number of xylose reductase (XR; encoding by XYL1 gene) on xylitol production of S. cerevisiae were investigated. The results showed that overexpression of XYL1 gene with a lower xylose-specificity and a higher NADPH preference favored the xylitol production. The copy number of XYL1 had a positive correlation with the XR activity but did not show a good correlation with the xylitol productivity. The overexpression of XYL1 from Candida tropicalis (CtXYL1) achieved a xylitol productivity of 0.83 g/L/h and a yield of 0.99 g/g-consumed xylose during batch fermentation with 43.5 g/L xylose and 17.0 g/L glucose. During simultaneous saccharification and fermentation (SSF) of pretreated corn stover, the strain overexpressing CtXYL1 produced 45.41 g/L xylitol and 50.19 g/L ethanol, suggesting its application potential for xylitol and ethanol coproduction from straw feedstocks.