Helicobacter pylori is an important risk factor of gastric cancer (GC). Although many Helicobacter pylori virulence factors have been reported, the pathogenic mechanism by which Helicobacter pylori infection causes GC remains unclear. The aims of this study are to identify GC-related antigens from Helicobacter pylori and characterize their roles in the development of GC. As GC and duodenal ulcer (DU) were considered clinical divergent, we compared two-dimensional immunoblots of an acid-glycine extract of Helicobacter pylori probed with serum samples from 15 patients with GC and 15 with DU to find GC-related antigens, which were subsequently identified by mass spectrometry. Many protein spots were recognized by more than one serum, and 24 of these were more recognized by GC. The proteins showing higher frequency of recognition in GC group are threonine synthase, rod shape-determining protein, S-adenosylmethionine synthetase, peptide chain release factor 1, DNA-directed RNA polymerase alpha subunit, co-chaperonin GroES (monomeric and dimeric forms), response regulator OmpR, and membrane fusion protein. Out of these proteins, GroES was identified as a dominant GC-related antigen, with a much higher seropositivity of GC samples (64.2%, n = 95) compared to 30.9% for gastritis (n = 94) and 35.5% for DU (n = 124). GroES seropositivity was more commonly associated with antral GC than with non-antral GC (odds ratio = 2.7; 95% confidence interval: 1.1-6.7). In PBMC, GroES stimulated production of IL-8, IL-6, granulocyte macrophage colony-stimulating factor, IL-1beta, TNF-alpha, cyclooxygenase-2, and prostaglandin E2. Moreover, when incubated with gastric epithelial cells, GroES induced expression of IL-8, cell proliferation and upregulation of c-jun, c-fos, and cyclin D1, but caused downregulation of p27Kip1. We conclude that GroES of H. pylori is a novel GC-associated virulence factor and may contribute to gastric carcinogenesis via induction of inflammation and promotion of cell proliferation.