BACKGROUND: As a quorum sensing system, LuxS/AI-2 is closely associated with bacterial growth, biofilm formation, and virulence. As yet, it is not known how the luxS is associated with a diverse array of physiological activities in non- carbapenemase producing carbapenem resistant Escherichia coli (non-CP-CREC). The purpose of this study is to explore the characterization of AI-2/LuxS quorum sensing system in antibiotic resistance, pathogenicity of non-CP-CREC. METHODS: A total of five non-CP-CREC isolates that did not have ompC and ompF deletions were collected from various clinical samples from January 2021 to December 2023. RT-qPCR was used to detect genes expression of luxS, acrA, acrB, tolC, mdtB, mdtC, mdtE, mdtF, ompA, ompX, IL-8, IL-6, and TNF-α. Homologous recombination was used to create the luxS knockout strain. Transcriptome sequencing was utilized to analyze gene expression changes before and after the luxS knockout. Biofilm formation was detected using crystal violet staining. Antimicrobial susceptibility test was used to determine drug resistance. Bacterial growth curves were used to detect the influence of the luxS on bacterial growth. A cell infection assay was used to detect the impact of the luxS on bacterial adhesion and the inflammatory response it induces. RESULTS: Our results indicated that the expression of the luxS was significantly elevated in non-CP-CREC strains compared to the carbapenem antibiotics sensitive E. coli (CSEC), with CREC229 exhibiting the most pronounced difference. Consequently, CREC229 was chosen for the development of the luxS knockout strain (CREC229△luxS). The deletion of the luxS did not impact the growth of non-CP-CREC. RNA sequencing analysis revealed that 82 genes were differentially expressed, with notable alterations observed in genes associated with biofilm formation regulation and outer membrane proteins in the ΔluxS strain. Our transcriptomic results show that the expression of bssS associated with biofilm formation is significantly reduced in the ΔluxS strain, which in turn reduces its capacity for biofilm formation. In addition, the luxS deletion increased the expression of adhesion-related genes, such as ompA and ompX, enhanced HCT-8 adherence to CREC229, and promoted the secretion of the inflammatory cytokine IL-6. In terms of bacterial resistance, the deletion of luxS increased the sensitivity of non-CP-CRECs to aminoglycoside antibiotics. CONCLUSIONS: LuxS/AI-2 quorum sensing systems can alter pathogenicity and resistance in several ways.