Helicobacter pylori is a gastric-dwelling pathogen responsible, with acid secretion, for peptic ulcer and a 20-fold increase in the risk of gastric cancer. Several transcriptomes have been described after short-term exposure to acidity in vitro, but there are no data identifying the effects of chronic gastric exposure on bacterial gene expression. Comparison of the in vivo to the in vitro transcriptome at pH 7.4 identified several groups of genes of known function that increased expression >2-fold, and three of these respond both to acidity in vitro and to gastric infection. Almost all known acid acclimation genes are highly up-regulated. These include ureA, ureB, and rocF and the pH-gated urea channel, ureI. There is also up-regulation of two groups of motility and chemotaxis genes and for pathogenicity island genes, especially cagA, a predictor for pathogenicity. Most of these genes interact with HP0166, the response element of the pH-sensing two-component histidine kinase, HP0165/HP0166, ArsRS. Based on the pH profile of survival of ureI deletion mutants in vitro and their inability to survive in gastric acidity, the habitat of the organism at the gastric surface is acidic with a pH ≤ 4.0. Hence, the pH of the habitat of H. pylori on the surface of the stomach largely determines the regulation of these specific groups of genes.