We have demonstrated that D-xylonate can be efficiently produced from D-xylose with Saccharomyces cerevisiae. 17±2 g D-xylonate l-1 at 0.23 g l-1 h-1 was produced from 23 g D-xylose l-1 (with glucose and ethanol as co-substrates) when expressing an NAD+-dependent D-xylose dehydrogenase, XylB, from Caulobacter crescentus. D-Xylonate titre and production rate were improved and xylitol production reduced, compared to strains expressing genes encoding Trichoderma reesei or pig liver NADP+-dependent D-xylose dehydrogenases. However, the production led to an intracellular accumulation of D-xylonate (up to 70 mg g-1) and xylitol (up to 18 mg g-1) and to a decreased viability of the D-xylonate producing cells. To reduce xylitol production, xylB was also expressed in a strain from which the major aldose reductase, encoded by GRE3, had been deleted. An industrial S. cerevisiae strain expressing XylB produced 43 g D-xylonate l-1 from 49 g D-xylose l-1, with an initial production rate of 0.44 g l-1 h-1.