Anticancer activities of soy isoflavones, such as genistein and equol, a bioactive metabolite of daidzein, have been extensively studied because of possible involvement in the prevention of breast cancer. However, their interactions still remain unclear. We investigated here whether cytotoxic activity of genistein was enhanced by equol, using estrogen receptor positive MCF-7, HER2-positive SK-BR-3, and triple-negative MDA-MB-468 cell lines. Although cytotoxicity of genistein did not significantly differ between three subtypes of breast cancer cells, cytotoxic activities of genistein were significantly enhanced in combination with 50M equol in MCF-7 cells, but not in SK-BR-3 and MDA-MB-468 cells. In fluorescence activated cell sorting (FACS) analyses, MCF-7 cells were arrested at the G2/M by genistein but at G1/S by equol. Combination treatment arrested cells at G2/M but abolished equol-induced G1 block, indicating an antagonistic activity of genistein against equol in cell-cycle progression. Although apoptosis was not so evident with genistein alone, the combination made a drastic induction of apoptosis, accompanied by the increase of Bax/Bcl-xL expression ratio, without affecting the activities of Akt and mTOR. Taken together, these data suggest that enhancement of genistein activity by equol would be mainly mediated by augmented induction of apoptosis rather than arrest or delay of the cell cycle.