Lysine crotonylation has attracted widespread attention in recent years. However, little is known about bacterial crotonylation, particularly crotonyltransferase and decrotonylase, and its effects on antibiotic resistance. Our study demonstrates the ubiquitous presence of crotonylation in E. coli, which promotes bacterial resistance to polymyxin. We identify the crotonyltransferase YjgM and its regulatory pathways in E. coli with a focus on crotonylation. Further studies show that YjgM upregulates the crotonylation of the substrate protein PmrA, thereby boosting PmrA’s affinity for binding to the promoter of eptA, which, in turn, promotes EptA expression and confers polymyxin resistance in E. coli. Additionally, we discover that PmrA’s crucial crotonylation site and functional site is Lys 164. These significant discoveries highlight the role of crotonylation in bacterial drug resistance and offer a fresh perspective on creating antibacterial compounds. Display omitted •YjgM functions as a crotonyltransferase in Escherichia coli•Crotonylation proteomics reveals differentially crotonylated proteins in PMB-R•YjgM promotes bacterial polymyxin resistance via the crotonylation of PmrA•Crotonylation promotes PmrA’s binding ability to the eptA promoter Zhuang et al. find that YjgM is a crotonyltransferase in Escherichia coli. YjgM improves the binding affinity between the polymyxin-resistant protein PmrA and the eptA promoter by increasing the crotonoylation level of PmrA, thereby encouraging the expression of eptA, resulting in the development of polymyxin resistance in E. coli.