In order to produce xylitol with high yield, experiments were carried out to develope xylitol dehydrogenase (XDH) defective m mutant from Pichia stipitis and to investigate the xylit이 fermentation characteristics of mutant strain. After treatment of P s stipitis with EMS, mutant PXM-4 was selected based on the XDH activity and xylitol production capability. Among the tested c cosubstrates, galactose was selected as an adequate cosubstrate on xyl뻐I production of mutant PXM-4. With the increase of galactose concentration, xylitol production was decreased because the transport of xylose into cell was inhibited by g galactose. The optimal concentration of galactose for the production of xylitol using 20 g/L xylose was 20 g/L. Under this c condition, maximum concentration of xylitol and yield were 14.4 g/L and 97%, respectively. In order to prevent the inhibitory e effect of xylose transport by galactose, galactose was fed with low concentration and the concentration of xylitol produced w was increased up to 25 g/L. In the fermentation of corn cob hydrolyzate by mutant PXM-4, xylose was completely converted t to xylit이 with a 100% yield in 4 days culture.