Zymomonas mobilis, a promising candidate for industrial biofuel production, is capable of nitrogen fixation naturally without hindering ethanol production. However, little is known about the regulation of nitrogen fixation in Z. mobilis. We herein conducted a high throughput analysis of proteome and protein acetylation in Z. mobilis under N2-fixing conditions and established its first acetylome. The upregulated proteins mainly belong to processes of nitrogen fixation, motility, chemotaxis, flagellar assembly, energy production, transportation, and oxidation-reduction. Whereas, downregulated proteins are mainly related to energy-consuming and biosynthetic processes. Our acetylome analyses revealed 197 uniquely acetylated proteins, belonging to major pathways such as nitrogen fixation, central carbon metabolism, ammonia assimilation pathway, protein biosynthesis, and amino acid metabolism. Further, we observed acetylation in glycolytic enzymes of central carbon metabolism, the nitrogenase complex, the master regulator NifA, and the enzyme in GS/GOGAT cycle. These findings suggest that protein acetylation may play an important role in regulating various aspects of N2-metabolism in Z. mobilis. This study provides new knowledge of specific proteins and their associated cellular processes and pathways that may be regulated by protein acetylation in Z. mobilis.