Estrogen receptor β (ERβ) is activated in the prostate by 5α-androstane-3β,17β-diol (3β-Adiol) where it exerts antiproliferative activity. The proliferative action of the androgen receptor is activated by 5α-dihydrotestosterone (DHT). Thus, prostate growth is governed by the balance between androgen receptor and ERβ activation. 3β-Adiol is a high-affinity ligand and agonist of ERβ and is derived from DHT by 3-keto reductase/3β-hydroxysteroid dehydrogenase enzymes. Here, we demonstrate that, when it is expressed in living cells containing an estrogen response element-luciferase reporter, 17β-hydroxysteroid dehydrogenase type 6 (17βHSD6) converts the androgen DHT to the estrogen 3β-Adiol, and this leads to activation of the ERβ reporter. This conversion of DHT occurs at concentrations that are in the physiological range of this hormone in the prostate. Immunohistochemical analysis revealed that 17βHSD6 is expressed in ERβ-positive epithelial cells of the human prostate and that, in prostate cancers of Gleason grade higher than 3, both ERβ and 17βHSD6 are undetectable. Both proteins were present in benign prostatic hyperplasia samples. These observations reveal that formation of 3β-Adiol via 17βHSD6 from DHT is an important growth regulatory pathway that is lost in prostate cancer.