Restriction of sulfur containing amino acids (SCAA) has been shown to elicit a similar increase in lifespan and decrease in age-related morbidity as caloric restriction. The singular importance of epithelial barrier function in both physiological homeostasis and prevention of inflammation raised the issue of examining the effect of SCAA restriction on epithelial tight junction structure and permeability. Using a well described in vitro, epithelial model, the LLC-PK1 renal epithelial cell line, the effects of SCAA restriction in culture medium were studied. Reduction of methionine by 90%, cysteine by 50% and total elimination of cystine resulted in dramatically lower intracellular pools of these amino acids and their metabolite, taurine, but the intracellular pools of the non-SCAA were all elevated. Cell growth and differentiation were maintained, and both confluent cell density and transepithelial short circuit current were unaffected. Certain tight junctional proteins such as occludin and claudins 1 and 2 were not altered. However, claudins 3 and 7 were significantly decreased in abundance, whereas claudins 4 and 5 were markedly increased in abundance. The functional result of these structural changes was improved barrier function as evidenced by increased transepithelial electrical resistance and decreased transepithelial (paracellular) diffusion of D-mannitol.