As a consequence of antibiotic overuse and misuse, nosocomial infections caused by multidrug-resistant bacteria represent an emerging health issue throughout the world. The prevalence of CTX-M-producing E. coli has been increasing in Asia. Outer membrane vesicles (OMVs) are released from the surfaces of many gram-negative bacteria which are consist of proteins, lipopolysaccharide(LPS), phospholipids, RNA, and DNA. In this study, we hypothesis that MDR plasmid plays important role in regulating host bacteria vesiculation, pathogenesis and metabolism. As a consequence, the proteomic profiles differ between OMVs from E. coli with and without MDR plasmid. Here, we identified 452 protein components of clinical isolated Escherichia coli derived OMVs. OMVs from wide type E. coli and cured strain yielded identifications of 451 and 315 proteins, respectively, with 314 proteins overlapping between the two strains, suggesting the important role of MDR plasmid in regulating vesiculation and cargo packaging. Besides,137 proteins were enriched in wide type E. coli OMV but not in OMV from cured strain. These unique proteins function in pathogenesis, nutrient acquisition and catalysis, shaping the character of OMV originate from bacteria with MDR plasmid. A comparative view of OMVs will help us not only to elucidate the biogenesis and functions of OMV from pathogenic bacteria with the presence of MDR plasmid, but also to develop vaccines and antibiotics against drug resistance strains.