Helicobacter pylori (H. pylori) is known to be a major pathogen causing gastric diseases through its direct localization in gastric epithelium cells. H. pylori releases outer membrane vesicles (OMVs) throughout the growth process. The content, function, and mechanism of H. pylori OMVs in gastric epithelial cells remain unclear. In this study, we extracted and characterized H. pylori OMVs of two strains (standard strain NCTC11637 and clinical strain Hp-400) and analyzed the specific content by proteomic technology. We identified more than 400 proteins in H. pylori OMVs. In addition, we investigated the impact of H. pylori OMVs on cellular functions by detecting proteomic changes in GES1 cells. GES1 cells cocultured with increasing concentrations of H. pylori OMVs were subjected to quantitative proteomic analyses using label-free methods for relative quantitation. The results showed that a total of 4261 proteins were verified, 153 of which were significantly altered in abundance when cocultured with NCTC11637 OMVs, and a total of 4234 proteins in Hp-400 OMVs, 390 of which were significantly altered. Gene ontology analysis and Kyoto encyclopedia of genes and genomes pathway mapping identified significantly altered inflammatory and cancer signaling pathways, including metabolic pathways and the PI3K-Akt signaling pathway. Furthermore, we explored the proteomic changes in GES1 cells induced by H. pylori. Bioinformatics analysis showed that changes in multiple pathways coincided with OMV-mediated proteomic changes. Based on these results, H. pylori induced pathogenicity in epithelial cells at least partially by secreting OMVs that mediated dramatic and specific proteomic changes in host cells. Data are available via ProteomeXchange with identifiers PXD025216, PXD025259, and PXD025281.© 2021 The Authors. Published by American Chemical Society.