Aims Acid exposure induces accumulation of certain metabolites in bacteria. The experimental objective was to identify the primary metabolites accumulating in Escherichia coli O26:H11 as a function of acid (pH 3 center dot 6) exposure. Methods and Results Conclusions Different buffers of pH 7 center dot 5 and 3 center dot 6 were used to study the metabolites accumulating in E. coli O26:H11 cells during such pH exposure. After 24 h of acid exposure, there was a 7-log decline in E. coli populations on trypticase soy agar plates. Untargeted metabolomic analysis identified 293 primary metabolites of which 145 metabolites were differentially (P < 0 center dot 01) accumulating between pH 7 center dot 5 and 3 center dot 6 in E. coli O26:H11. After 24 h of acid exposure, 21 different metabolic pathways appeared to be functional, suggesting that the cells were still metabolically active. Among the identifiable pathways, the key differentially expressed pathways were associated with peptidoglycan biosynthesis, purine metabolism, d-Glutamine/d-glutamate metabolism, nitrogen metabolism, unsaturated fatty acid biosynthesis and inositol phosphate metabolism. Significance and Impact of the Study Shiga toxin producing non-O157 E. coli strains such as E. coli O26 are responsible for a growing number of food-related illnesses in the United States and around the world. From food production to consumption, micro-organisms in foods experience dramatic pH fluctuations by organic acids introduced either during food processing or by inorganic acids in the stomach. Acid exposure induces specific metabolite accumulation in bacterial cells. Understanding the survival mechanisms of pathogenic micro-organisms by studying the metabolome would be helpful in introducing effective hurdles and thus ensuring food safety.