Infection with Helicobacter pylori in the gastric mucosa is thought to be increased the risk of peptic ulcer disease and gastric cancer. Vacuolating cytotoxin A (VacA) was an important bacterial virulence factor and was closely associated with Helicobacter pylori-induced pathogenesis. Upon Helicobacter pylori infection, the VacA can be transferred directly from bacteria into the host cells where can stimulate vacuolar-type ATPase (V-ATPase) proton pump and induce the formation of vacuoles in gastric epithelial cells followed by intoxication of cells. Destruxin B (Dtx-B), a cyclodepsipeptide from the fungus Metarhizium anisopliae bearing insecticidal and anticancer effects. In this study, Dtx-B demonstrated inhibitory effects against Helicobacter pylori VacA-induced vacuolization in gastric epithelial cells. Our data further showed that attenuation of Helicobacter pylori-induced vacuolization in gastric epithelial cells by Dtx-B is mediated through inhibition of V-ATPase activity rather than VacA expression levels. The results from this study reveal a novel role for Dtx-B in the modulation of Helicobacter pylori VacA-induced pathogenesis. (C) 2014 Elsevier Ltd. All rights reserved.