Gram-negative bacterial lipopolysaccharide (LPS) increases the susceptibility of cells to pathogenic diseases including inflammatory diseases and septic syndrome. In our experiments, we examined whether LPS induces epithelial barrier disruption in secretory epithelia and further investigated the underlying mechanism. The activities of Ca2+-activated Cl- channels (CACC) and epithelial Na+ channels (ENaC) were monitored with a short-circuit current using an Ussing chamber. Epithelial membrane integrity was estimated via transepithelial electrical resistance and paracellular permeability assays. We found that apical application of LPS evoked Isc through the activation of CACC and ENaC. LPS disrupted epithelial barrier integrity, but this was restored with the inhibition of CACC and ENaC. Notably, CACC or ENaC activation as well as exposure to a high K+ buffer evoked apical membrane depolarization and increased paracellular permeability, indicating the role of CACC and ENaC in the regulation of paracellular pathways. LPS induced the rapid redistribution of zonula occludens-1 (ZO-1) and reduced the expression levels of ZO-1 in tight junctions through apical membrane depolarization and tyrosine phosphorylation. However, the LPS-induced epithelial barrier disruption and degradation of ZO-1 were largely recovered by blocking CACC and ENaC. Furthermore, LPS-impaired epithelial barrier became vulnerable to secondary bacterial infections and this vulnerability was also prevented by the inhibition of CACC and ENaC. We concluded that LPS induces the disruption of epithelial barrier integrity through the activation of CACC and ENaC, resulting in apical membrane depolarization and subsequent tyrosine phosphorylation of ZO-1.