The intestinal barrier serves 2 critical functions for the survival of the individual: first, it allows nutrient absorption and second, it defends the body from dangerous macromolecule penetration. It is a complex multilayer system, consisting of an external “anatomic” barrier and an inner “functional” immunological barrier. The interaction of these 2 barriers enables equilibrated permeability to be maintained. Many factors can alter this balance: gut microflora modifications, mucus layer alterations, and epithelial damage can increase intestinal permeability, allowing the translocation of luminal content to the inner layer of intestinal wall. Several techniques are now available that enable us to study gut permeability: “in vitro” models (Caco-2 and HT29-MTX cells) and “in vivo” not invasive tests (sugar tests and radioisotope scanning tests) are used to estimate permeability and to suggest molecular pathophysiological mechanisms of intestinal permeability in health and diseases. Many medicinal products used in the treatment of gastrointestinal diseases have also found to play an active role in modulate intestinal permeability: corticosteroids, 5-aminosalicylic acid, anti–tumor necrosis factor, probiotics, and mucosal protectors, like gelatin tannate. This review will particularly address the role of the gut barrier in maintaining intestinal permeability (microbiota, mucus, and epithelial cells), the techniques used for estimating intestinal permeability and the therapeutic approaches able to modify it.