Helicobacter pylori vacuolating toxin (VacA) is a secreted toxin that is reported to produce multiple effects on mammalian cells. In this study, we explored the relationship between VacA-induced cellular vacuolation and VacA-induced cytochrome c release from mitochondria. Within intoxicated cells, vacuolation precedes cytochrome c release and occurs at lower VacA concentrations, indicating that cellular vacuolation is not a downstream consequence of cytochrome c release. Conversely, bafilomycin A1 blocks VacA-induced vacuolation but not VacA-induced cytochrome c release, which indicates that cytochrome c release is not a downstream consequence of cellular vacuolation. Acid activation of purified VacA is required for entry of VacA into cells, and correspondingly, acid activation of the toxin is required for both vacuolation and cytochrome c release, which suggests that VacA must enter cells to produce these two effects. Single amino acid substitutions (P9A and G14A) that ablate vacuolating activity and membrane channel-forming activity render VacA unable to induce cytochrome c release. Channel blockers known to inhibit cellular vacuolation and VacA membrane channel activity also inhibit cytochrome c release. These data indicate that cellular vacuolation and mitochondrial cytochrome c release are two independent outcomes of VacA intoxication and that both effects are dependent on the formation of anion-selective membrane channels.