Abstract: To investigate the role of puuC, which encodes γ-glutamyl-γ-aminobutyraldehyde dehydrogenase, in bacterial antibiotic tolerance and its potential as a novel target for combating antibiotic resistance, we constructed an Escherichia coli puuC-overexpressing strain（BW25113-puuC）and used a wild-type strain carrying an empty plasmid（BW25113-pCA24N）as the control. Killing assays with gentamicin and kanamycin, minimum inhibitory concentration（MIC）determination, and growth curve analysis were performed. We further compared the tolerance of the puuC-overexpressing strain to β-lactam and quinolone antibiotics. The results showed that overexpression of puuC significantly increased the survival of E. coli under gentamicin and kanamycin treatment without increasing the MIC, indicating tolerance rather than resistance. Moreover, the puuC-overexpressing strain exhibited some tolerance to β-lactam antibiotics but no obvious tolerance to quinolones. These findings suggest that puuC mainly mediates E. coli tolerance to aminoglycoside antibiotics, and that the putrescine metabolic pathway in which its encoded enzyme participates may serve as a potential intervention target for strategies to combat bacterial antibiotic resistance.