Debaryomyces hansenii is one of the most promising natural xylitol producers. As the conversion of xylitol to xylulose mediated by NAD(+) cofactor dependent xylitol dehydrogenase (XDH) reduces its xylitol yield, xylitol dehydrogenase gene (DhXDH)-disrupted mutant of D. hansenii having potential for xylose assimilating pathway stopping at xylitol, was used to study the effects of co-substrates, xylose and oxygen availability on xylitol production. Compared to low cell growth and xylitol production in cultivation medium containing xylose as the only substrate, XDH disrupted mutants grown on glycerol as co-substrate accumulated 2.5-fold increased xylitol concentration over those cells grown on glucose as co-substrate. The oxygen availability, in terms of volumetric oxygen transfer coefficient, kLa (23.86-87.96 h(-1)), affected both xylitol productivity and yield, though the effect is more pronounced on the former. The addition of extra xylose at different phases of xylitol fermentation did not enhance xylitol productivity under experimental conditions.