Helicobacter pylori infection, pepsin, ischemia, hypoxia, smoking, alcohol intake, hydrochloric acid, and nonsteroidal anti-inflammatory drugs are the causal factors of ulceration. In third-world countries, approximately one in two individuals has signs of gastric ulceration. Extensive literature suggested that plant-derived drugs hold the potential in the treatment of peptic ulcer, and Rheum spiciforme (family: Polygonaceae) has been known in the folk medicine for possessing medicinal effect on ulceration. To explore further along this line, the current study was designed to investigate the effects of aqueous ethanol extract of R. spiciforme roots and its fractions on peptic ulcer and to identify bioactive compounds responsible for mitigating this pathological condition. Animal model of ethanol-induced gastric ulcer was utilized in this study. The animals were pre-treated with crude plant extract, given at doses 125, 250, and 500 mg/kg, and then orally administered with butanol, aqueous, and dichloromethane fractions at doses of 250 mg/kg for 7 days. Following the treatments, a significant decrease in ulcerative lesions, ulcer index, ulcer severity score, volume, free acidity, and total acidity of gastric juice as well as a marked increase of gastric pH in the groups treated with plant extract at doses of 250 and 500 mg/kg were noted. Moreover, butanol fraction has been shown to produce effects equipotent to that of the reference drug omeprazole. Liquid chromatography-mass spectrometry analyses show that the plant extract contains emodin, aloe-emodin, and quercetin. In conclusion, this study demonstrated that the aqueous ethanol extract of R. spiciforme and its butanol fraction exhibited gastro-protective effect.