Eupatorium adenophorum can induce liver toxicity in animals. For the safe utilisation of the weed, the hepatotoxic components need to be discovered. In this study, in vitro hepatotoxicity of different extracts from E. adenophorum were determined on human hepatocyte cell line L02 and hepatocellular carcinoma cell line HepG2. The results showed that water extracts of E. adenophorum exhibited no hepatotoxicity in vitro while high concentrations of the organic solvent extracts had obvious hepatotoxicity. Sesquiterpenes may contribute to the toxicity based on the comparison of composition analysis. Three cadinene sesquiterpenes were purified and identified as 9-oxo-10,11-dehydroageraphorone, 10H alpha-9-oxo-ageraphorone and 10H alpha-9-oxoageraphorone. In vitro hepatotoxic effects of these components were investigated, the IC50 of the three compounds were 122.53, 87.52, and 108.80 mu M in L02 cells and 151.92, 104.48, and 138.08 mu M in HepG2 cells by Cell Counting Kit-8 (CCK-8) assay. The three components were confirmed to be, at least partial, hepatotoxic components.