The mechanisms that maintain the epithelial integrity of the gastrointestinal tract remain largely undefined. The gene encoding intestinal trefoil factor (ITF), a protein secreted throughout the small intestine and colon, was rendered nonfunctional in mice by targeted disruption. Mice lacking ITF had impaired mucosal healing and died from extensive colitis after oral administration of dextran sulfate sodium, an agent that causes mild epithelial injury in wild-type mice. ITF-deficient mice manifested poor epithelial regeneration after injury. These findings reveal a central role for ITF in the maintenance and repair of the intestinal mucosa.