Abstract: The molecular mechanism involved in tolerance and adaptation of ethanologenic yeast to inhibitors (such as furfural, acetic acid and phenol) represented in lignocellulosic hydrolysate is still unclear. Here, (18)O labeling-aided shotgun comparative proteome was applied to study the global protein expression profiles of S. cerevisiae under the treatment of furfural compared with the furfural-free fermentation. Proteins involved in glucose fermentation and/or TCA cycle were upregulated in cells treated with furfural compared with the control cells, while proteins involved in glycerol biosynthesis were downregulated. The differential expressions of alcohol dehydrogenases were observed. On the other hand, the levels of NADH, NAD(+) and NADH/NAD(+) were reduced while the level of ATP/ADP was increased. These observations indicated that central carbon metabolism, alcohol dehydrogenases and the redox balance may be related to the tolerance and adaptation of ethanologenic yeast to furfural. Furthermore, proteins involved in stress response, including unfolded protein response, oxidative stress, osmotic and salt stress, DNA damage and nutrient starvation were differentially expressed, which were validated by quantitative RT-PCR to further confirm that the general stress response are essential for cellular defense against furfural. These insights into the response of yeast to furfural will benefit the design and development of inhibitor-tolerant ethanologenic yeast by metabolic engineering or synthetic biology.