The intestinal barrier is not a passive membrane but a layered and actively regulated interface. The epithelium and its tight junctions form the physical layer, which is complemented by a chemical layer of mucus and antimicrobial peptides, by the mucosal immune compartment, and by the resident microbiota. Together these layers separate a substantial microbial and dietary antigen load from the systemic circulation. Zonulin, identified as pre-haptoglobin-2, has been implicated in the reversible regulation of intestinal tight junctions while the broader concept of zonulin-related proteins (ZRP) encompasses proteins potentially associated with this regulatory system. Loss of barrier control has an established role in the pathogenesis of inflammatory bowel disease and has also been described in obesity, insulin resistance and atherosclerosis, with emerging evidence of an association with cardiovascular outcomes. Circulating ZRP have consequently attracted interest as potential biomarkers of intestinal barrier dysfunction and associated inflammatory and metabolic disturbances. However, uncertainty regarding the molecular specificity of commercial immunoassays complicates their biological interpretation and warrants further analytical and clinical validation. In this Review, we place zonulin within barrier physiology, appraise the clinical evidence in these disorders, and set out the analytical limitations that constrain its use as a biomarker.