Bioconversion of undetoxified hemicellulosic hydrolyzate from steam-exploded corn stover was investigated with a domesticated Pichia stipitis CBS 5776. The countercurrent washing was applied to recover sugars from the steam-exploded corn stover, which could enrich sugars in washing liquor and give an efficient saving of water. Acid concentration, reaction temperature, and time were optimized for the acid post-hydrolysis of oligosaccharides in steam-exploded prehydrolyzate by a central composite design and response surface methodology. The domestication of P. stipitis to the hydrolyzate resulted in improving sugar consumption and ethanol yield by gradually increasing the ratio of hydrolyzate in the medium. Recycling utilization of the domesticated yeast demonstrated that the yeast kept a stable ability of fermenting both hexose and pentose in the undetoxified hydrolyzate. The sugar consumption and ethanol yield were over 90 and 80 %, respectively.