Tetragenococcus halophilus is known to flourish in extreme salt environments, and frequently existed during the manufacture of fermented foods, where various environmental stresses are encountered. In this study, acid tolerance response of T. halophilus was investigated based on physiological and proteomic analysis. The optimal acid-adaptation condition was optimized to stimulate acid tolerance response effectively. After acid adaptation at pH 4.0 for 1 h, the highest survival rate at lethal pH 2.5 was observed. Analysis of the physiological data showed that higher intracellular pH and NH4+ pool were detected in cells adapted at pH 4.0 compared with cells without acid adaptation. In addition, the activities of hexokinase, pyruvate kinase, and lactate dehydrogenase were determined, and significantly higher activities were monitored in cells after acid adaptation. Comparison of the intracellular amino acid pools indicated that six amino acids (glutamate, aspartate, isoleucine, leucine, citrulline, ornithine) exhibited notable alternations during acid treatments, and acid pre-adaption led to higher pools of amino acid compared to that without acid pre-treatment. Proteomic analysis demonstrated that eight proteins (Glk, Pfk, Fba, Gap, Gpm, Eno, Pyk, and Ldh) involved in carbohydrate metabolism and three classic stress response proteins (Ssb, UspA, and GroL) were up-regulated after acid adaptation. Results presented in this study may help to further understand the acid tolerance response in T. halophilus and improve the industrial performance of this species. (C) 2015 Elsevier Ltd. All rights reserved.