ABSTRACT Mucosal barriers separate self from non-self and are essential for life. These barriers, which are the first line of defense against external pathogens, are formed by epithelial cells and the substances they secrete. Rather than an absolute barrier, epithelia at mucosal surfaces must allow selective paracellular flux that discriminates between solutes and water while preventing the passage of bacteria and toxins. In vertebrates, tight junctions seal the paracellular space; flux across the tight junction can occur through two distinct routes that differ in selectivity, capacity, molecular composition and regulation. Dysregulation of either pathway can accompany disease. A third, tight-junction-independent route that reflects epithelial damage can also contribute to barrier loss during disease. In this Cell Science at a Glance article and accompanying poster, we present current knowledge on the molecular components and pathways that establish this selectively permeable barrier and the interactions that lead to barrier dysfunction during disease.