Background and Objective: Calea urticifolia is used in traditional medicine to treat gastric ulcer but there has not as yet been any scientific report on its therapeutic effects. The aim of this study was tested the gastroprotective activity of C. urticifolia. Materials and Methods: The gastroprotective activity of C. urticifolia was evaluated using bioassay-guided fractionation and the model of ethanol-induced gastric lesions in Wistar rats. The statistical significance between treatments was evaluated by the Kruskal-Wallis test, followed by Dunn’s multiple comparison test. Results: The results show that C. urticifolia has gastroprotective activity. The dichloromethane extract was the most active, yielding 95.7±1.5% gastroprotection with a dose of 30 mg kg–1. Five fractions were obtained from the dichloromethane extract and 2,3-epoxyjuanislamin was isolated from one of them. Rats treated with 2,3-epoxyjuanislamin at doses of 3, 10 and 30 mg kg–1 presented 64.63±7.46, 83.03±4.63 and 96.1±1.59% gastroprotection, respectively. The effect of 2,3-epoxyjuanislamin at 30 mg kg–1 was not modified by pretreatment with indomethacin, NG-nitro-L-arginine methyl ester or N-ethylmaleimide. Conclusion: The bioassay-guided study of C. urticifolia validates of the traditional use of this plant for gastric ulcers treatment. The 2,3-epoxyjuanislamin, isolated from the dichloromethane extract was identified as one of the compounds responsible for this activity and its mechanism of action is apparently not related to prostaglandins, nitric oxide or sulfhydryl groups.