The Trapa natans L. has been believed rich in nutritional compositions for a long time. The purpose of this study was to investigate the anti-proliferation activity and action mechanism of the ethanol extract of Trapa natans L. to MDA-MB-231 cells. Firstly, from the MTT assay the ethanol extract of Trapa natans L. showed the great anti-proliferation activity to MDA-MB-231 cells. Therefore, we focused study the action mechanism of the ethanol extract of Trapa natans to MDA-MB-231 cells. The result showed the ethanol extract of Trapa natans L. made the cell ladders of MDA-MB-231; increased the cell populations at the Sub-G1 phase; finally decreased the intracellular ROS; and increased the expression of Bad; decreased Bcl-2 and pro-caspase 9.