Gentianae Radix, the dried root and rhizoma of Gentiana lutea L. (Gentianaceae), has long been used as a remedy for liver and stomach inflammation, eye troubles, etc. In this paper, the gastroprotective effects of the methanol extract of Gentian root (GM) were studied using different gastric lesion models. In pylorus-ligated rats, administration of GM in the duodenum suppressed gastric juice secretion and total acid output in a dose-dependent manner. Oral or duodenum administration of GM showed significant protection against acute gastric ulcer induced by aspirin plus pylorus ligation, water-immersion restraint stress-induced ulcers, and gastric mucosal injury induced by ethanol. Furthermore, four secoiridoid glycosides, amarogentin (A1), gentiopicroside (A2), amaroswerin (A3), and swertiamarin (A4), were obtained from Gentian root or Swertia herb, and their protective effects against stress-induced ulcers and ethanol-induced gastric mucosal injury were evaluated. The doses required for 50% inhibition (ID50) of A1, A3, and A4 on stress-induced ulcers were calculated to be 5.76, 2.58, and 167mg/kg respectively. The protective effect of A2at 250mg/kg was 26.5%. On ethanol-induced gastritis, 5.0mg/kg of A1 and A3 showed remarkable suppressive effects (33.7 and 45.4%, respectively), and 20mg/kg of A4 exhibited a suppressive effect (30.8%). The effects of A1, A3, and A4 on ethanol-induced gastric lesions were canceled by 5.0mg/kg indomethacin pretreatment. These results suggest that the therapeutic effects of Gentian root on gastric lesions are associated with enhanced mucosal defensive factors via the prostaglandin pathway in the cell membrane, and that secoiridoid glycosides contribute to this activity.