Acetic acid and furfural are the two prevalent inhibitors coexisting with glucose and xylose in lignocellulosic hydrolysate. The transcriptional regulations of S. cerevisiae in response to acetic acid (Aa), furfural (Fur), and the mixture of acetic acid and furfural (Aa_Fur) while fermenting with glucose and xylose were revealed. The pathways classified as carbohydrate metabolism were significantly enriched in response to Aa, while the pathways belonged to xenobiotics biodegradation and metabolism were significantly enriched in response to Fur. In addition to these pathways, some new pathways were activated in response to Aa_Fur, i.e., cofactors and vitamins metabolism, and lipid metabolism. Overexpression of Haa1p or Tye7p improved xylose consumption rate by nearly 50%, while the ethanol yield enhanced by nearly 8%. Further co-overexpression of Haa1p and Tye7 resulted in 59% increase in xylose consumption rate and 12% increase in ethanol yield, revealing the beneficial effects of Haa1p and Tye7p on improving the tolerance of yeast to mixed fermentation inhibitors.