Intestinal ischemia results in mucosal injury, including paracellular barrier loss due to disruption of tight junctions. Larazotide acetate (LA), a small peptide studied in Phase III clinical trials for treatment of celiac disease, regulates tight junctions (TJs). We hypothesized that LA would dose-dependently hasten recovery of intestinal ischemic injury via modulation of TJs. Ischemia-injured tissue from 6-8-week-old pigs was recovered in Ussing chambers for 240-minutes in the presence of LA. LA (1 μM but not 0.1 μM or 10 μM) significantly enhanced transepithelial electrical resistance (TER) above ischemic injured controls and significantly reduced serosal-to-mucosal flux LPS (P<0.05). LA (1 μM) enhanced localization of the sealing tight junction protein claudin-4 in repairing epithelium. To assess for the possibility of fragmentation of LA, an in vitro enzyme degradation assay using the brush border enzyme aminopeptidase M, revealed generation of peptide fragments. Western blot analysis of total protein isolated from uninjured and ischemia-injured porcine intestine showed aminopeptidase M enzyme presence in both tissue types, and mass spectrometry analysis of samples collected during ex vivo analysis confirmed formation of LA fragments. Treatment of tissues with LA fragments had no effect alone, but treatment with a fragment missing both amino-terminus glycines inhibited barrier recovery stimulated by 1 μM LA. To reduce potential LA inhibition by fragments, a D-amino acid analog of larazotide Analog #6, resulted in a significant recovery response at a 10-fold lower dose (0.1 μM) similar in magnitude to that of 1 μM LA. We conclude that LA stimulates repair of ischemic-injured epithelium at the level of the tight junctions, at an optimal dose of 1 μM LA. Higher doses were less effective because of inhibition by LA fragments, which could be subverted by chirally-modifying the molecule, or microdosing LA.