Antibiotic-resistant Vibrio alginolyticus becomes a worldwide challenge threatening both human health and food safety. The approach in managing such infection is largely absent, despite the fact that the mechanisms of antibiotic resistance have been extensively investigated. Metabolic modulation has been documented to be a novel approach in improving antibiotic efficacy. In this study, we characterize the metabolic signature of V. alginolyticus exposed to 0.3 or 0.5 μg/ml of ofloxacin (OFX). By profiling the metabolome, we find that bacteria treated by the two different concentrations of OFX generate different metabolic signatures. While a part of these metabolites was shared by both groups, the other metabolites represent their own signatures. The pathway enrichment analysis demonstrates that the pyruvate cycle is disrupted in the bacteria treated by the 0.3 μg/ml OFX as compared to those by the 0.5 μg/ml. Importantly, the disruption of pyruvate cycle confers the capability of bacteria to survive under 0.5 μg/ml of antibiotic stress. Further analysis identifies that the fatty acid biosynthesis is elevated in bacteria treated by 0.3 μg/ml OFX, and inhibition on fatty acid completely prevents the bacteria from survival even under such dose of antibiotic stress. Our study suggests that bacteria adapt to antibiotic stress by modulating the metabolic flux for survival, which could be targeted to increase antibiotic efficacy.