A better understanding of normal prostate epithelial stem cells may lead to new ways to target prostate cancer repopulating cells. The prostate is exquisitely sensitive to hormones; castration causes normal prostate tissue to regress, but surviving epithelial stem cells completely regenerate the gland when testosterone is restored. We now show that regeneration can be impaired by activating estrogen receptor β (ERβ) in normal, castrate-resistant stem cells. Unlike multiple cycles of castration and testosterone replacement, where the prostate fully regenerates, successive treatments of adult mice with a specific ERβ agonist, 8βVE2, caused a progressive loss of prostate tissue architecture. To verify that this effect was due to the action of 8βVE2 on regenerative stem cells, 8βVE2 was administered to adult mice 10 days after castration. This induced a second wave of apoptosis in castrate-resistant cells that ordinarily regenerate the prostate when testosterone is restored. In addition, 8βVE2 treatment attenuated the clonogenicity and self renewal of primary prostate spheroids in vitro. We further hypothesised that the ability of 8βVE2 to inhibit normal prostate regeneration could be applied to prostate cancer. First, we developed a model of cancer repopulating cells. Like the normal mouse prostate, a subset of tumour cells in xenografts of localised human prostate cancer survived castration. These “castrate tolerant” cells exhibited the characteristics of cancer repopulating cells, expressing prostate stem cell markers and regenerating the tumour when testosterone was restored. Therefore, in ongoing experiments the ability of ERβ agonists to target the castrate tolerant cells is being assessed. Collectively, these results show that insights into the biology of normal prostate stem cells, such as their susceptibility to ERβ activation, can be translated into potential treatments for prostate cancer.