Two flavonoids, genistein and apigenin, have been implicated as chemopreventive agents against prostate and breast cancers. However, the mechanisms behind their respective cancer-protective effects may vary significantly. The goal of this study was to determine whether the antiproliferative action of these flavonoids on prostate (DU-145) and breast (MDA-MB-231) cancer cells expressing only estrogen receptor (ER) β is mediated by this ER subtype. It was found that both genistein and apigenin, although not 17β-estradiol, exhibited anti proliferative effects and proapoptotic activities through caspase-3 activation in these two cell lines. In yeast transcription assays, both flavonoids displayed high specificity toward ERβ transactivation, particularly at lower concentrations. However, in mammalian assay, apigenin was found to be more ERβ-selective than genistein, which has equal potency in inducing transactivation through ERα and ERβ. Small interfering RNA-mediated downregulation of ERβ abrogated the anti proliferative effect of apigenin in both cancer cells but did not reverse that of genistein. Our data unveil, for the first time, that the anticancer action of apigenin is mediated, in part, by ERβ. The differential use of ERα and ERβ signaling for transaction between genistein and apigenin demonstrates the complexity of phytoestrogen action in the context of their anticancer properties.