The vacuolating toxin VacA, a major determinant of Helicobacter pylori‐associated gastric diseases, forms anion‐selective channels in artificial planar lipid bilayers. Here we show that VacA increases the anion permeability of the HeLa cell plasma membrane and determines membrane depolarization. Electrophysiological and pharmacological approaches indicated that this effect is due to the formation of low‐conductance VacA pores in the cell plasma membrane and not to the opening of Ca 2+‐or volume‐activated chloride channels. VacA‐dependent increase of current conduction both in artificial planar lipid bilayers and in the cellular system was effectively inhibited by the chloride channel blocker 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB), while2‐[(2‐cyclopentenyl‐6, 7dichloro‐2, 3‐dihydro‐2‐methyl‐1‐oxo‐1H‐inden‐5‐yl) oxy] acetic acid (IAA‐94) was less effective. NPPB inhibited and partially reversed the vacuolation of HeLa cells and the increase of ion conductivity of polarized Madine Darby canine kidney cell monolayers induced by VacA, while IAA‐94 had a weaker effect. We conclude that pore formation by VacA accounts for plasma membrane permeabilization and is required for both cell vacuolation and increase of trans‐epithelial conductivity.