VacA toxin is one of the most important virulence factors produced by H. pylori even though neither its role nor its action mechanisms are completely understood. First considered as a toxin inducing only cell vacuolation, VacA causes apoptosis of gastric epithelial cells by targeting mitochondria. A hotly debated question about VacA action is its relationship with ammonia, which is produced in vivo by H. pylori urease. While ammonia is strictly required for VacA-dependent vacuolation, its role in VacA-induced apoptosis is much less defined. This study was thus aimed to investigate the relationship between VacA toxin and ammonia in H. pylori-induced mitochondrial damage and apoptosis of human gastric epithelial cells in culture by means of flow cytometry. Our results show that, unlike cell vacuolation, in MKN 28 cells neither apoptosis nor dissipation of mitochondrial transmembrane potential induced by VacA require ammonia. Nevertheless, ammonia significantly potentiates both these VacA-induced effects, but independently of the swelling of VacA-containing endosomes (i.e., vacuolation). Our findings make unlikely the hypothesis that ammonia-dependent swelling and rupture of endosomal vesicles in which VacA is sequestered after cell internalization may allow the toxin to reach mitochondria and trigger apoptosis.