Zinc homoeostasis exerts protective effects in inflammatory intestinal diseases and zinc supplementation has been successfully used for treating infectious diarrhoea. This study aimed at a characterisation of zinc effects on focal leak induction by α-haemolysin (HlyA)-producing Escherichia coli (E. coli) as protective mechanism for colitis. We conducted in vivo experiments by oral challenge of gnotobiotic mice colonised with HlyA-expressing E. coli-536. Mice were either fed a defined normal or high zinc diet to analyse effects of zinc as a therapeutic regimen. HlyA-deficient E. coli-536 mutants were used as controls. Mice infected with HlyA-producing E. coli showed impaired barrier integrity when receiving normal zinc. High zinc supplementation in HlyA-producing E. coli-infected mice reduced epithelial dysfunction as indicated by ameliorated macromolecule permeability. Reduced size of focal leaks with diminished bacterial translocation was observed as inherent mechanisms of this zinc action. In human colon cell monolayers application of zinc rescued the HlyA-dependent decline in transepithelial electrical resistance via reduction of the calcium entry into HlyA-exposed cells. Calcium-dependent cell exfoliation was identified as mechanism for focal leak induction. In conclusion, zinc supplementation protects from HlyA-induced barrier dysfunction in vivo and in vitro, providing an explanation for the protective efficacy of zinc in intestinal disorders.