Escherichia coli O157:H7 has an unusually high resistance to acidic environments. Some research has revealed that acid-adapted cells, by exposure to moderately acidic conditions, are more resistant to a subsequent strong acidic challenge or other stress. This study was conducted to understand the protein expression regulation of acid tolerance response (ATR) of a local isolated E. coli O157:H7 TWC01 (TWC01) induced by an acidic environment. TWC01 cells were acid adapted by using hydrochloric acid (HCl) or lactic acid as acidifier to induce ATR. The total proteins of adapted cells were extracted for proteomic analysis and protein identification by matrix-assisted laser desorption ionization quadrupole time-of-flight tandem mass spectrometry (MALDI-Q-TOF MS/MS). Furthermore, the effects of acid adaptation on shiga-like toxin (stx) secretion were examined. Results revealed that acid adaptation depressed stx production of E. coli O157:H7 TWC01 during adaptation and did not improve post-stress toxin production. Image analysis of the gel indicated that numerous proteins were up-regulated and that lactic acid had a greater effect than HCl did (percentages of up-regulated proteins were 57.64 and 35.47%, respectively). Analysis of proteins by mass spectrometry revealed that most of the up-regulated proteins were metabolism-related, including phosphoglycerate kinase (PGK), glutamate decarboxylases alpha and beta (GadA, GadB), adenine phosphoribosyltransferase (APRT), and dihydrodipicolinate synthase (DHDPS). Others were related to translation (e.g., elongation factor Tu, elongation factor G), protein folding (e.g., alkyl hydroperoxide reductase), and membrane proteins (e.g., ompA precursor and ompR). The variation of protein expression showed that acid resistance was induced in TWC01 and was primarily manifested via expression of up-regulated proteins that contribute to increased energy conservation and polypeptide synthesis.