Yersinia enterocolitica is a pathogenic microorganism that can cause food-borne diseases. Lipoic acid (LA) has been used as an antioxidant against bacteria, but its antibacterial mechanism is rarely reported. This study aims to explore the antibacterial mechanism of LA and its effect on the metabolites of Y. enterocolitica through membrane damage and metabolomics analysis. The results showed that the minimum inhibitory concentration (MIC) of LA against Y. enterocolitica was 2.5 mg mL-1. The membrane potential was depolarized, and intracellular pH (pHin) and ATP were significantly reduced, indicating that LA destroys the cell membrane structure. Confocal laser scanning microscopy (CLSM) and field emission scanning electron microscopy (FESEM) further confirmed LA-induced cell membrane damage. The metabolic profile of Y. enterocolitica following LA treatment was analyzed by liquid chromatography-mass spectrometry (LC-MS). In the metabolome evaluation, 6209 differential metabolites were screened, including 3394 up-regulated and 2815 down-regulated metabolites. Fifteen metabolic pathways of Y. enterocolitica exhibited significant changes after LA treatment, including the pathways important for amino acid and nucleotide metabolism. The results show that LA is a bacteriostatic substance with potential application value in the food industry.