Updated publication reference for PubMed record(s): 31551951. Protein phosphorylation is an important post translational modification that plays a major role in cellular regulatory processes in both eukaryotes and prokaryotes. In recent years, aided by advancements in mass spectrometry techniques, there has been a growing interest in studying protein phosphorylation in prokaryotic model organisms. There are, however, only a limited number of phosphoproteomics reports on non-model organisms. Here, using mass spectrometry, we performed a genome wide investigation of protein phosphorylation in the non-model organism and biofuel producer Zymomonas mobilis under anaerobic, aerobic, and N2-fixing conditions. Our phosphoproteome analysis revealed 125 unique phosphorylated proteins and 172 unique phosphopeptides across these three growth conditions. The phosphoproteins identified belonged to major pathways, including glycolysis, TCA cycle, protein biosynthesis, electron transport, and nitrogen fixation. Quantitative analysis revealed significant and widespread changes in protein phosphorylation across anaerobic, aerobic, and N2-fixing growth conditions. For example, two different serine residues of KDPG aldolase, an Entner-Doudoroff pathway enzyme, were differentially phosphorylated under aerobic and N2-fixing conditions. On the other hand, the final enzyme in the ethanol fermentation pathway, alcohol dehydrogenase, showed phosphorylation on its Ser99 only under N2-fixing condition while its Ser126 was only phosphorylated under anaerobic condition. Moreover, nitrogenases and nitrogen regulatory proteins were differentially phosphorylated at multiple sites under aerobic and N2-fixing conditions. Altogether, this study provides new knowledge regarding potential phosphorylation regulatory sites of specific proteins in pathways relevant to ethanol production and overall physiology and establishes new ground for future engineering of Z. mobilis for advanced biofuel production.