Ingestion of contaminated dairy products, in particular soft cheese, is one of the major routes of infection by the human pathogen Listeria monocytogenes. During cheese processing, this foodborne pathogen is exposed to sublethal acid and osmotic stress conditions, which may induce tolerance responses and influence subsequent survival in the gastric tract. The aim of the current study was to evaluate the impact on a L. monocytogenes cheese isolate (serotype 4b) and two cheese dairy isolates (T8, serotype 4b, isolated from vat; and A9, serotype 1/2b or 3b, isolated from shelf stand) of exposure to sublethal conditions of pH and salt (5.5 and 3.5% [w/v] NaCl) in a cheese-simulated medium and further challenge with gastric stress. The bacterial cells exposed to pH 7.0 and no added salt were considered non-adapted. Via two-dimensional gel electrophoresis (2-DE), the proteomes of cheese-simulated medium and gastric challenged Listeria cells were compared. All L. monocytogenes isolates were able to survive the high acidity of gastric fluid (pH 2.5), and no significant differences were observed between adapted and non-adapted cells. However, the analysis of the intracellular proteome profiles revealed a significant intra-strain variation in the protein arsenal used to respond to the adaptation in the cheese-based medium and to the gastric stress. In cheese-based medium, the three strains produced different stress proteins. All three strains showed a higher abundance of carbohydrate proteins, but there was no overlap between them. Exposure to the gastric fluid induced the production of a group of proteins in T8 adapted and non-adapted cells that had not been detected previously in the cheese-based proteome. No such response was shown by A9 and C882 strains. Taken together, this study evidences the proteome tools used by adapted and non-adapted cells to cope with the hostile microenvironment of the stomach.