The objective of the present study was to determine if a specific window of development (neonatal/ prepubertal only, adult only, or life-time) is effective for genistein chemoprevention of prostate cancer, and the potential mechanisms of genistein chemoprevention in vivo. Male Lobund-Wistar (L-W) rats were fed zero or 250 mg genistein/kg AIN-76A diet at designated periods of time and then injected with N-methylnitrosourea (NMU) into the dorsolateral prostate (DLP) on day 70 for cancer initiation. Rats were necropsied at 11 months. The incidence of poorly differentiated (PD) carcinomas was 43.5% in rats fed a phytoestrogen-free AIN-76A diet only, 29.6% in rats provided genistein in the diet from postnatal days 1-35, 28.6% in rats fed genistein from months 3-11, and 20% in rats provided genistein from birth through 11 months. Genistein induces cell apoptosis and inhibits cell proliferation in both prostate cancerous and nontumorigenic DLP. These actions are accompanied with the regulation of PTEN/Akt-AR axis. Our data demonstrate that dietary genistein reduces the incidence of advanced prostate cancer induced by NMU in L-W rats during adult and life-time exposure, the latter being more effective. The regulation of AR/Akt/PTEN axis by genistein may be one of the molecular mechanisms by which it inhibits cell proliferation and induces apoptosis, thus providing evidence of roles of genistein in prostate cancer prevention and treatment.