Hesperidin, a flavonoid derived from citrus fruits, has been reported to show various biological effects including anticancer activity. This study investigated whether hesperidin affected the proliferation of MCF-7 human breast cancer cells transfected with green fluorescent protein (GFP)/alpha-tubulin (MCF-7-GFP-Tubulin cells), androgen-independent PC-3 and DU-145 prostate cancer cells, and androgen-dependent LNCaP prostate cancer cells. The results were as follows. (1) Hesperidin inhibited the proliferation of MCF-7-GFP-Tubulin cells, probably not through an antimitotic mechanism. (2) Hesperidin also inhibited both basal and testosterone-induced proliferation of LNCaP cells. (3) However, hesperidin did not significantly affect the cell proliferation of two hormone-independent prostate cancer cells, PC-3 and DU-145. It is concluded that hesperidin can inhibit the proliferation of breast cancer cells through mechanisms other than antimitosis and it is suggested that hesperidin be further investigated for the possible interaction with androgenic receptors and involvement in signaling pathway after receptor binding in prostate cancer cells through future research.