Helicobacter pylori colonization of the gastric mucosa induces peptic ulcer disease and interferes with ulcer healing. Re-epithelialization is an essential component of ulcer healing. It requires cell migration and proliferation which are dependent on the cell cytoskeleton. Most H. pylori strains produce a toxin (VacA) that induces multiple structural and functional changes in epithelial cells. In this study, we investigated the effects of VacA on the gastric epithelial cell cytoskeletal architecture. Exposure of rat gastric epithelial cells to purified VacA from H. pylori 60190 significantly inhibited actin stress fiber formation (83 ± 5% reduction; p < 0.0001) and disorganized microtubule pattern (90 ± 8%; p < 0.001). Furthermore, VacA treatment significantly reduced tyrosine phosphorylation of focal adhesion kinase (FAK) (by 45 ± 6%; p < 0.002) and its expression in focal adhesions (73 ± 8%; p < 0.0001). These findings suggest that H. pylori VacA interferes with cytoskeleton-dependent cell functions and with the transmission of signals related to cell spreading and growth.