Lobelia giberroa Hemsl. is an indigenous plant in Ethiopia. Ethiopian traditional healers use its rndifferent parts to treat malaria, bacterial and fungal infections, and cancer. For treatment of rnmalaria, in particular, the traditional healers utilize the plant’s root parts. This study was carried rnout to investigate the antimalarial activity of L. giberroa. rnThe roots of L. giberroa were extracted using 80% methanol and the hydroalcoholic extract was rnsuccessively fractionated with hexane, ethyl acetate, methanol and water. Chromatographic and rnspectroscopic methods were used to isolate and identify the active compound from the methanol rnfraction. Acute oral toxicity study was conducted on the hydroalcoholic extract, solvent rnfractions, sub-fractions and the isolated compound. The hydroalcoholic extract and the isolated rncompound were evaluated for antimalarial activity using the standard four-day suppressive rnmethod, Rane’s and prophylactic tests in Plasmodium berghei infected albino mice. The solvent rnfractions and sub-fractions from column chromatography were evaluated for antimalarial activity rnusing four-day suppressive method. rnThe hydroalcoholic extract, solvent fractions, subfractions and the isolated compound were rnfound to be safe at a dose of 2000 mg/kg. The hydroalcoholic extract of L. giberroa root and its rnmethanol fraction exhibited the highest antimalarial activity and significantly increased the mean rnsurvival time of the treated mice. In fact, the hydroalcoholic extract exhibited 73.05%, 49.35% rnand 43.16% parasitemia suppression for four-day suppressive test, Rane's test and prophylactic test, respectively. The methanol fraction demonstrated 64.37% parasitemia suppression for four-rnday suppressive test. rnThe isolated compound, characterized as lobetyolin, suppressed the level of parasitaemia by rn39.96, 53.46 and 68.21% at a dose of 25, 50 and 100 mg/kg, in standard four-day suppressive rntest, respectively. Mice treated with 100 mg/kg/day lobetyolin survived longer (18.6 days) when rncompared with negative control group (6.8 days). rnIn conclusion, the current study supports the traditional use of the plant for the treatment of rnmalaria and identified the main active compound lobetyolin, the potential to be an antimalarial rnlead for further development. The active ingredient, lobetyolin, was also isolated for the first rntime from the root of Lobelia giberroa. To the best of our knowledge, this is the first report on rnthe biological activity of L. giberroa.