Industrial production of 2-keto-L-gulonic acid (2-KLG), the precursor of vitamin C, is mainly achieved by a two-step fermentation process carried out byGluconobacter oxydans,Bacillus, andKetogulonicigenium. One of the most promising innovations that could replace this complicated two-step fermentation process is the integration of the essential genes for synthesis of 2-KLG intoG. oxydansand use of it as the producer. Therefore, determining the tolerance and response ofG. oxydansto 2-KLG is a priority for improving the direct production of 2-KLG in this bacterium. In this study, a global view of the gene expression ofG.oxydansWSH-003 in response to 2-KLG challenge was investigated by RNA sequencing. A total of 363 genes ofG. oxydansthat were differentially expressed in response to 2-KLG were uncovered. The results showed that 2-KLG could lead to oxidative stress, osmotic stress, and DNA damage inG. oxydans.