One of the most important and overlooked functions of the gastrointestinal tract is to provide a dynamic barrier to tightly controlled antigen trafficking through both the transcellular and paracellular pathways. Intercellular tight junctions (TJ) are the key structures regulating paracellular trafficking of macromolecules. Although steady progress has been made in understanding TJ ultrastructure, relatively little is known about their pathophysiological regulation. Our discovery of zonulin, the only known physiological modulator of intercellular TJ described so far, increased understanding of the intricate mechanisms that regulate gut permeability and led us to appreciate that its up-regulation in genetically susceptible individuals may lead to immune-mediated diseases. This information has translational implications, because the zonulin pathway is currently exploited to develop both diagnostic and therapeutic applications pertinent to a variety of immune-mediated diseases.