Helicobacter pylori possesses intrabacterial nanotransportation systems (ibNoTSs) for CagA and urease. Both systems are UreI-dependent and urea-independent, and activated by extrabacterial acid. The activation occurs/appears within 15 min after exposure to extrabacterial acid stimulation. Although it has been clarified that VacA is secreted via the type-V secretion machinery, it remains unclear how this toxin is transported toward the machinery. To clarify the intrabacterial nanotransportation system for H. pylori VacA, immunoelectron microscopic analysis was performed in this study. VacA shifted to the periphery of the bacterial cytoplasm at 30 min after the extracellular pH change, whereas CagA and urease did so within 15 min. Studies using an ureI-deletion mutant revealed that unlike CagA and urease transport, VacA transport was not UreI-dependent. VacA secretion was accelerated without an increase in the production of VacA 30 min after the exposure to extrabacterial acid. These findings indicated that H. pylori possesses a novel type of ibNoTS for VacA, which is different from that for CagA or urease, in response time and dependency of UreI.