ABSTRACT Bifidobacteria are one of the main microbial inhabitants of the human colon. Usually administered in fermented dairy products as beneficial microorganisms, they have to overcome the acidic pH found in the stomach during the gastrointestinal transit to be able to colonize the lower parts of the intestine. The mechanisms underlying acid response and adaptation in Bifidobacterium longum biotype longum NCIMB 8809 and its acid-pH-resistant mutant B. longum biotype longum 8809dpH were studied. Comparison of protein maps, and protein identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, allowed us to identify nine different proteins whose production largely changed in the mutant strain. Furthermore, the production of 47 proteins was modulated by pH in one or both strains. These included general stress response chaperones and proteins involved in transcription and translation as well as in carbohydrate and nitrogen metabolism, among others. Significant differences in the levels of metabolic end products and in the redox status of the cells were also detected between the wild-type strain and its acid-pH-resistant mutant in response to, or as a result of, adaptation to acid. Remarkably, the results of this work indicated that adaptation and response to low pH in B. longum biotype longum involve changes in the glycolytic flux and in the ability to regulate the internal pH. These changes were accompanied by a higher content of ammonium in the cytoplasm, likely coming from amino acid deamination, and a decrease of the bile salt hydrolase activity.