Tight junctions are widely present between epithelial cells and endothelial cells, which can regulate the transport of substances through paracellular pathways and maintain cell polarity. Among them, the tight junctions between intestinal epithelial cells can prevent various antigens from leaking into the blood circulation and maintain the body’s homeostasis, the disruption of which may lead to a variety of intestinal and extraintestinal diseases. The elevated level of zonulin, an intestinal epithelial tight junction opener, serves as an early event in intestinal diseases such as celiac disease. Larazotide acetate (also known as AT-1001), a competitive inhibitor of zonulin, has been shown by in vitro studies and clinical trials to effectively stabilize the structure of tight junction complexes, enhance barrier functions, and alleviate the occurrence and development of diseases caused by the breakdown of tight junction barrier functions. This review focuses on the mechanism of action of AT-1001 and its application in the treatment of various diseases such as celiac disease, providing a comprehensive understanding of the future clinical application of AT-1001.