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The dichloromethane extract of the stem bark of Millettia usaramensis subspecies usaramensis showed anti-plasmodial activity against the chloroquine sensitive (D6) and chloroquine resistant (W2) strains of Plasmodium falciparum. Chromatographic separation of the extract led to the identification of a new rotenoid, (6aR,12aS)-2,3-methylenedioxy-9-methoxy-8-(3,3-dimethylallyl)-12a-hydroxyrotenoid (trivial name, usararotenoid C) along with known flavonoids (usararotenoid A, 12a-epimillettosin, 6a,12a-dehydromillettone, barbigerone and 4′-O-geranylisoliquiritigenin) as the anti-plasmodial principles. The structures were determined by spectroscopic analyses. CD and X-ray analyses established absolute configurations.