Helicobacter pylori infection is a crucial factor in the pathogenesis of several digestive disorders, including peptic ulcers, chronic gastritis, and gastric cancer. Moreover H. pylori induces disease-specific protein expression in gastric epithelial cells. The aim of the present study was to characterize proteins differentially expressed in H. pylori-infected gastric epithelial AGS cells. An in vitro model was established using a multiplicity of infection of 100 and evaluating the effectiveness of H. pylori infection by functional analyses. Changes in protein patterns were identified using a proteomic approach consisting of two-dimensional fluorescence difference gel electrophoresis and mass spectrometry. The expression of many proteins was found to be altered, and 28 of these were identified and classified as protein synthesis- and folding-related proteins, cytoskeleton proteins, metabolic enzymes, transcription- and translation-related proteins, angiogenesis/metastasis-related proteins, cell communication/signal transduction-related proteins, or others (oxygen-regulated protein and oncoprotein). The expression profiles of eight of these proteins, laminin gamma-1 chain precursor, valosin-containing protein, heat shock 70-kDa protein, mitochondrial matrix protein P1, FK506-binding protein 4, T-complex protein 1, enolase alpha, and 14-3-3 beta were further examined in cancerous and paired surrounding normal tissues by immunoblot assay and immunohistochemical staining to identify molecular targets that may be involved in the pathogenesis of H. pylori-induced gastric diseases. On the basis of our results, valosin-containing protein, mitochondrial matrix protein P1, T-complex protein 1, enolase alpha, and 14-3-3 beta may play a crucial role in H. pylori-induced gastric carcinogenesis by mediating antiapoptotic and proliferative responses.