The intestinal epithelium, the largest interface between the host and environment, regulates fluxes of ions and nutrients and limits host contact with the massive load of luminal antigens. Local protective and tolerogenic immune responses toward luminal content depend on antigen sampling by the gut epithelial layer. Whether, and how exaggerated, the entrance of antigenic macromolecules across the gut epithelium might initiate and/or perpetuate chronic inflammation as well as the respective contribution of paracellular and transcellular permeability remains a matter of debate. To this extent, experimental studies involving the in vivo assessment of intestinal permeability using small inert molecules do not necessarily correlate with the uptake of larger dietary antigens. This review analyzes both the structural and functional aspects of intestinal permeability with special emphasis on antigen handling in healthy and diseased states and consequences on local immune responses to food antigens.