Intestinal mucositis is a frequent side effect of chemotherapy with 5-fluorouracil, characterized by a gastrointestinal inflammatory process. Epiisopiloturine is an imidazole alkaloid from Pilocarpus microphyllus Stapf ex Wardlew., Rutaceae, considered promising due to its high economic potential, since it is a by-product from the isolation of pilocarpine. In addition, this drug has antioxidant and anti-inflammatory activities. This study aimed to investigate the effects of epiisopiloturine on 5-fluorouracil-induced intestinal mucositis. After extraction, epiisopiloturine was characterized by the application of chromatographic, spectroscopic, and thermal methods. In the in vivo study, the animals received a single 5-fluorouracil dose (450 mg/kg, i.p.) and, from the second to the fifth day, were treated with epiisopiloturine (0.1, 1 or 10 mg/kg, i.p.). After euthanasia, the intestinal samples were removed for evaluation of morphometric and histopathological scores, the number of mast cells, goblet cells, circulating leukocytes, and the concentration of malondialdehyde and glutathione. Finally, we evaluated the expression of cyclooxygenase-2 by immunohistochemistry. Epiisopiloturine presented anti-inflammatory action through the reduction of inflammation indicators and oxidative stress. Epiisopiloturine also preserved the number of circulating leukocytes, contributed to the recovery of the tissue architecture, and restored the number of goblet cells in the small intestine. Thus, our findings suggest that epiisopiloturine protects the intestinal mucosa through inhibition of COX-2.