Intestinal ischemia is associated with high morbidity and mortality, but the underlying mechanisms are uncertain. We hypothesize that during ischemia the intestinal mucosal barrier becomes disrupted, allowing digestive enzymes access into the intestinal wall initiating autodigestion. We used a rat model of splanchnic ischemia by occlusion of the superior mesenteric and celiac arteries up to 30 min with and without luminal injection of tranexamic acid as a trypsin inhibitor. We determined the location and activity of digestive proteases on intestinal sections with in situ zymography, and we examined the disruption of two components of the mucosal barrier: mucin isoforms and the extracellular and intracellular domains of E cadherin with immunohistochemistry and Western blot techniques. The results indicate that nonischemic intestine has low levels of protease activity in its wall. After 15-min ischemia, protease activity was visible at the tip of the villi, and after 30 min, enhanced activity was seen across the full thickness of the intestinal wall. This activity was accompanied by disruption of the mucin layer and loss of both intracellular and extracellular domains of E cadherin. Digestive protease inhibition in the intestinal lumen with tranexamic acid reduced morphological damage and entry of digestive enzymes into the intestinal wall. This study demonstrates that disruption of the mucosal epithelial barrier within minutes of intestinal ischemia allows entry of fully activated pancreatic digestive proteases across the intestinal barrier triggering autodigestion.