The ability of exogenous low ouabain concentrations to affect claudin expression and therefore epithelial barrier properties was demonstrated previously in cultured cell studies. We hypothesized that chronic elevation of circulating ouabain in vivo can affect the expression of claudins and tight junction permeability in different tissues. We tested this hypothesis in rats intraperitoneally injected with ouabain (1 μg/kg) for 4 days. Rat jejunum, colon and brain frontal lobes, which are variable in the expressed claudins and tight junction permeability, were examined. Moreover, the porcine jejunum cell line IPEC-J2 was studied. In IPEC-J2-cells, ouabain (10 nM, 19 days of incubation) stimulated epithelial barrier formation, increased transepithelial resistance and the level of cSrc-kinase activation by phosphorylation, accompanied with an increased expression of claudin-1, -5 and down-regulation of claudin-12; the expression of claudin-3, -4, -8 and tricellulin was not changed. In the jejunum, chronic ouabain increased the expression of claudin-1, -3 and -5 without an effect on claudin-2 and -4 expression. In the colon, only down-regulation of claudin-3 was observed. Chronic ouabain protected the intestine transepithelial resistance against functional injury induced by lipopolysaccharide treatment or by modeled acute microgravity; this regulation was most pronounced in the jejunum. Claudin-1 was also up-regulated in cerebral blood vessels. This was associated with reduction of claudin-3 expression while the expression of claudin-5 and occludin was not affected. Altogether, our results confirm that circulating ouabain can functionally and tissue-specifically affect barrier properties of epithelial and endothelial tissues via Na,K-ATPase-mediated modulation of claudins expression.