During the post weaning period, piglets are prone to gastrointestinal infections. The resulting impairment of intestinal barrier function may cause diarrhea associated with growth retardation or even death of piglets. Orally applied zinc is commonly used to prevent and treat diarrhea but its mode of action still needs to be elucidated. In order to analyze the molecular mechanism whereby zinc acts on porcine intestinal barrier function, ex vivo studies on piglet jejunum and accompanying in vitro studies on a porcine jejunal epithelial cell line, IPEC-J2/PS, were performed with electrophysiological tools. Feeding pharmacological zinc doses exerted no significant electrophysiologically ascertainable short- and long-term effects on jejunal barrier function ex vivo. However, in IPEC-J2/PS, basolateral zinc was cytotoxic since its application caused a release of lactate dehydrogenase and an irreversible break-down of the epithelial barrier. In contrast, apical zinc application caused an immediate increase in paracellular resistance and a decrease in permeability to the paracellular marker fluorescein, reflecting overall barrier strengthening in vitro. Apical effects were fully reversible upon wash-out. This indicates that zinc supplemented to feed was completely washed out during ex vivo jejunum preparation. We conclude that there is no evidence for long-term barrier effects through prophylactic zinc supplementation and that extracellular zinc acts acutely and reversibly from the apical side via tightening the paracellular route, thus counteracting leak-flux diarrhea.