Metabolic reprogramming mediates antibiotic efficacy. However, metabolic adaptation of microbes evolving from antibiotic sensitivity to resistance remains undefined. Therefore, untargeted metabolomics was conducted to unveil relevant metabolic reprogramming and potential intervention targets involved in gentamicin resistance. In total, 61 metabolites and 52 metabolic pathways were significantly altered in gentamicin-resistant E. coli . Notably, the metabolic reprogramming was characterized by decreases in most metabolites involved in carbohydrate and amino acid metabolism, and accumulation of building blocks for nucleotide synthesis in gentamicin-resistant E. coli . Meanwhile, fatty acid metabolism and glycerolipid metabolism were also significantly altered in gentamicin-resistant E. coli . Additionally, glycerol, glycerol-3-phosphate, palmitoleate, and oleate were separately defined as the potential biomarkers for identifying gentamicin resistance in E. coli . Moreover, palmitoleate and oleate could attenuate or even abolished killing effects of gentamicin on E. coli , and separately increased the minimum inhibitory concentration of gentamicin against E. coli by 2 and 4 times. Furthermore, palmitoleate and oleate separately decreased intracellular gentamicin contents, and abolished gentamicin-induced accumulation of reactive oxygen species, indicating involvement of gentamicin metabolism and redox homeostasis in palmitoleate/oleate-promoted gentamicin resistance in E. coli . This study identifies the metabolic reprogramming, potential biomarkers and intervention targets related to gentamicin resistance in bacteria.