Helicobacter pylori contributes to the development of peptic ulcers and atrophic gastritis. Furthermore, H. pylori strains carrying the cagA gene are more virulent than cagA-negative strains and are associated with the development of gastric adenocarcinoma. The cagA gene product, CagA, is translocated into gastric epithelial cells and localizes to the inner surface of the plasma membrane, in which it undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motif. Tyrosine-phosphorylated CagA specifically binds to and activates Src homology 2-containing protein-tyrosine phosphatase-2 (SHP-2) at the membrane, thereby inducing an elongated cell shape termed the hummingbird phenotype. Accordingly, membrane tethering of CagA is an essential prerequisite for the pathogenic activity of CagA. We show here that membrane association of CagA requires the EPIYA-containing region but is independent of EPIYA tyrosine phosphorylation. We further show that specific deletion of the EPIYA motif abolishes the ability of CagA to associate with the membrane. Conversely, reintroduction of an EPIYA sequence into a CagA mutant that lacks the EPIYA-containing region restores membrane association of CagA. Thus, the presence of a single EPIYA motif is necessary for the membrane localization of CagA. Our results indicate that the EPIYA motif has a dual function in membrane association and tyrosine phosphorylation, both of which are critically involved in the activity of CagA to deregulate intracellular signaling, and suggest that the EPIYA motif is a crucial therapeutic target of cagA-positive H. pylori infection.