Cases of human African trypanosomiasis caused by infection with a protozoan parasite, Trypanosoma brucei, are decreasing due to enhanced surveillance and control. However, effective and safe treatments for this disease are still needed. In this study, we investigated the antitrypanosomal activity of citrus fruit peel. When 19 citrus cultivars were examined for activity against T. brucei in vitro, significant activities were observed in four closely related cultivars and a distantly related one. Among these five cultivars, "Setoka" was selected for identification of its active components due to exhibiting the highest activity. Solvent extraction and gel filtration followed by preparative thin-layer chromatography succeeded in isolating two compounds exhibiting ICs of 4.8 and 2.4 μg/mL, respectively. The spectral data of these two compounds were well consistent with those of sinensetin and nobiletin belonging to the class of polymethoxyflavones. Authentic compounds also showed similar ICs. These results indicate that the two polymethoxyflavones are the major active components involved in the inhibition of T. brucei proliferation and are abundant in Setoka cultivar peel compared with the levels in the other cultivars. Setoka peel and the naturally occurring polymethoxyflavones might serve as dietary components imparting resistance to T. brucei.