Euphorbia helioscopia L is considered a traditional Chinese herb which is widely distributed in China. The active anticancer fractions and anticancer mechanism of the herb are unclear. In this study, we evaluated the growth inhibitory effects of Euphorbia helioscopia L extracts on five different human cancer cell lines for screening the main active fraction with antitumor effect. In this regard, the ethyl acetate extract (EAE) was found to markedly inhibit the proliferation of SMMC-7721 cells in a time- and dose-dependent manner. EAE treatment arrested cell cycle in G-1 phase and EAE used at the concentration range of 100–200 μg/mL induced a marked increase of subdiploid peak. After EAE treatment at the concentrations of 150 and 200 μg/mL, the percentage of apoptotic cells was increased. At the EAE concentration of 200 μg/mL, the typical morphology of early apoptotic change was observed in SMMC-7721 cells. Since tumorigenesis is often defined by an uncontrolled proliferation and transplantability, we also determined the anti-invasive effects of EAE. The EAE treatment displayed a dose-dependent inhibitory effect on tumor cell invasion and MMP-9 expression. Also, the major active fraction was assayed using high-performance liquid chromatography (HPLC). The data showed that the flavonoids could be the main constituents of EAE. Based on the evidence from these data, we inferred that the EAE of Euphorbia helioscopia L could have chemopreventive potential against the human cancer. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.