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) beta is mediated by this ER subtype. It was found that both genistein and apigenin, although not 17beta-estradiol, exhibited antiproliferative effects and proapoptotic activities through caspase-3 activation in these two cell lines. In yeast transcription assays, both flavonoids displayed high specificity toward ERbeta transactivation, particularly at lower concentrations. However, in mammalian assay, apigenin was found to be more ERbeta-selective than genistein, which has equal potency in inducing transactivation through ERalpha and ERbeta. Small interfering RNA-mediated downregulation of ERbeta abrogated the antiproliferative 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 ERbeta. The differential use of ERalpha and ERbeta signaling for transaction between genistein and apigenin demonstrates the complexity of phytoestrogen action in the context of their anticancer properties.