Abstract The development of inhibitor-tolerant ethanologenic yeast is one of the most significant challenges facing bio-ethanol production. Adaptation of Pichia stipitis to inhibitors is one of the most efficient ways for dealing with inhibitor problems. The molecular mechanisms involved in the tolerance and adaptation of P. stipitis are, however, still unclear. In the present study, we developed a yeast strain from P. stipitis Y7 that has improved tolerance against inhibitors. We performed comparative proteomic investigations with sodium dodecyl sulfate polyacrylamide gel electrophoresis and quadrupole time-of-flight mass spectrometry. These investigations gave insights into the tolerance of yeast strains to biomass conversion inhibitors at the protein level. Many proteins involved in glycolysis, the pentose phosphate pathway, and the tricarboxylic acid (TCA) cycle were found to be differentially expressed due to the presence of furfural. Quantitative real-time reverse transcription-PCR (RT-PCR) and metabolite analysis were utilized to provide orthogonal evidence for the results obtained. Our results provide a deeper understanding of the molecular mechanisms involved in the response of P. stipitis to furfural. These findings will benefit the design and development of inhibitor-tolerant yeast.