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Oral insulin (INS) has been shown to protect intestinal epithelial cells from injury caused by ischemia-reperfusion and endotoxemia. In the present study, we tested whether oral insulin can protect gut epithelial cells from methotrexate (MTX)-induced intestinal damage. Adult male Sprague-Dawley rats were divided into 3 experimental groups. Control rats were treated with normal saline given intraperitoneally (CONTR), MTX rats were treated with a single dose (20 µg/kg) of MTX given intraperitoneally, and MTX-INS rats were treated with oral insulin given in drinking water (1 U/mL) 72 hours after IP injection of a single dose of MTX (similar to MTX rats). Three days after either MTX or saline injection, rats were killed. Intestinal mucosal damage (Park injury score), mucosal structural changes, enterocyte proliferation, and enterocyte apoptosis were measured. Reverse transcription polymerase chain reaction was used to determine the level of bax and bcl-2 mRNA expression. MTX-INS rats demonstrated a greater jejunal and ileal mucosal weight, ileal mucosal DNA, greater jejunal villus height, greater jejunal and ileal crypt depth, greater enterocyte proliferation index in ileum, and lower enterocyte apoptosis in ileum than did MTX-nontreated animals. Treatment with insulin did not change the injury score grade in comparison with MTX animals. A significant decrease in cell apoptosis was observed in MTX-INS rats (vs MTX) and also a decrease in a bax mRNA expression and decrease in a bax/bcl-2 ratio. In a rat model of MTX-induced mucositis, oral insulin supplementation does not prevent mucosal injury but improves intestinal recovery and enhances enterocyte survival.