Survival of bacteria in acidic environments is critical for pathogens to successfully multiply and infect the gastrointestinal tract. In this study, a metabolomics approach was conducted to understand and explain the effect of pH-induced stress metabolites in Yersinia enterocolitica. Cells were cultured in tryptic soy broth with or without pH adjustment using HCl (pH 6.8 or pH 3, respectively). In this study, we employed untargeted proton nuclear magnetic resonance (1H-NMR) spectroscopy followed by statistical multivariate analysis was used to identify the metabolites and measure the metabolic profiles and pathways impacted by acid stress. Under conditions of low pH, increased branched-chain amino acid biosynthesis and protein acetylation were predominantly observed. There was also evidence of osmotic dysregulation during acid challenge. Modulation of these systems might confer bacteria with resistance to stressors encountered during infection of organisms or food contamination. The study presented here with other related researches may help to explain further, how pathogenic bacteria survive and tolerate the environmental stresses.