BACKGROUND Flos Genkwa (yuanhua in Chinese), the dried flower buds of Daphne genkwa Sieb.et Zucc. (Thymelaeaceae), is a traditional Chinese medicinal herb mainly used for diuretic, antitussive, expectorant, and anticancer effects. However, systematic and comprehensive studies on Flos Genkwa and its bioactivity are limited. RESULTS After confirmation of the anti-tumor activity, the 95% ethanolic extract was subjected to successive solvent partitioning to petroleum ether, dichloromethane, n-butanol, and water soluble fractions. Each fraction was tested using the same biological activity model, and the dichloromethane fraction had the highest activity. The dichloromethane fraction was subjected to further chromatographic separation for the isolation of compounds 1-13. Among the 13 compounds, the diterpene esters (compounds 10-13) showed anticancer activity, whereas the flavonoids, lignanoids, and peptides showed moderate activity. Compound 13 was a new daphnane diterpenoid, which was named genkwanin VIII.The preliminary antitumor mechanism of yuanhuacine was studied by protein expression and cell cycle analysis in MCF-7 cancer cells. CONCLUSIONS The present investigation tends to support the traditional use of Flos Genkwa for treating cancer. Through bioassay-guided fractionation and isolation techniques, the CH2Cl2 fraction was determined as the active fraction of the flower buds of D. genkwa, and the anti-tumor activity was ascribable to the compounds 10-13.