Baker's yeast number 6 was selected by screening. It showed good catalytic activity and enantioselectivity for asymmetric reduction of 2,5-hexanedione to produce (2S,5S)-2,5-hexanediol. Gas chromatography-mass spectrometry (GC-MS) revealed that the intermediate was (S)-5-hydroxyhexane-2-one. Reduction of 2,5-hexanedione proceeded in a two-step reaction. The hydroxyketone was initially formed, and this intermediate was further reduced to the diol. Factors influencing the product yield and the enantiomeric excess of the reduction of 2,5-hexandione catalyzed by baker's yeast number 6 were investigated. Higher concentration (<= 100 mmol.L-1) of 2,5-hexandione did not influence 5-hydroxyhexane-2-one production, but 2,5-hexanediol production was inhibited by excess accumulation (>30 mmol.L-1) of intermediate. The optimal conditions were glucose as the co-substrate at an initial glucose concentration of 20 g.L-1, 34 degrees C, pH 7.0 and cell concentration 60 g.L-1 (cell dry mass). Under the optimal condition and an initial substrate concentration of 30 mmol.L-1, the yield of 2,5-hexandiol was 78.7% and the enantiomeric excess of (2S,5S)-2,5-hexandiol was 94.4% for 24-h reduction.