2-Phenylethanol (2-PE) is a kind of advanced aromatic alcohol with rose fragrance, which is wildly used for the deployment of flavors and fragrances. Microbial transformation is the most feasible method for the production of natural 2-PE. But a bottleneck problem is the toxicity of 2-PE on the cells. The molecular mechanisms of the toxic effect of 2-PE to Saccharomyces cerevisiae are not well studied. In this study, we analyzed the transcriptomes of S. cerevisiae in the media with and without 2-PE, respectively, using Illumina RNA-Seq technology. We identified 580 differentially expressed genes between S. cerevisiae in two different treatments. GO and KEGG enrichment analyses of these genes suggested that most genes encoding mitochondrial proteins, cytoplasmic, and plasma membrane proteins were significantly up-regulated, whereas the enzymes related to amino acid metabolism were down-regulated. These results indicated that 2-PE suppressed the synthesis of plasma membrane proteins, which suppressed the transport of nutrients required for growth. The findings in this study will provide insight into the inhibitory mechanism of 2-PE to yeast and other microbes.