Prostate cancer is one of the most prevalent malignant cancers in men. The isoflavone formononetin is a mainactive component of red clover plants. In the present study, we assessed the effect of formononetin on humanprostate cancer DU-145 cells in vitro, and elucidated posssible mechanisms. DU-145 cells were treated withdifferent concentrations of formononetin and cell proliferation was assessed by MTT assay, cell apoptosis byHoechst 33258 and flow cytometry, and protein levels of RASD1, Bcl-2 and Bax by Western blotting. The resultsshowed that formononetin inhibited the proliferation of DU-145 cells in a dose-dependent manner. DU-145 cellstreated with different concentrations of formononetin displayed obvious morphological changes of apoptosisunder fluorescence microscopy. In addition, formononetin increased the proportion of early apoptotic DU-145cells, down-regulated the protein levels of Bcl-2 and up-regulated those of RASD1 and Bax. The level of RASD1reached its maximum at 48h post-treatment, and rapidly decreased thereafter. Together, we present evidence thatformononetin triggered cell apoptosis through the mitochondrial apoptotic pathway by up-regulating RASD1.