Abstract: To address the issue of antibiotic resistance and elucidate the potential function of the argC gene in Escherichia coli, particularly its role in the formation of antibiotic tolerance, this study evaluated the tolerance phenotype by measuring the survival rate of an argC knockout strain（ΔargC）under ampicillin treatment. Non-specific physiological effects were excluded through growth curve analysis. Further tests examined the tolerance phenotype of ΔargC to various antibiotics, including amikacin（aminoglycoside）, ciprofloxacin（quinolone）, and ceftriaxone（β-lactam）. The results showed that compared to the wild-type strain, ΔargC exhibited a significantly higher survival rate under ampicillin treatment, and this phenotype was not caused by growth delay. Additional broad-spectrum antibiotic testing revealed that ΔargC developed tolerance to amikacin, ciprofloxacin, and other antibiotics but showed no tolerance to ceftriaxone. These findings indicate that the deletion of the argC gene can mediate the development of tolerance to multiple antibiotics in E. coli, providing new theoretical insights and research directions for understanding resistance evolution pathways and developing novel infection control strategies.