Estrogens play a pivotal role in the development and progression of prostate cancer (PCa). Their actions are mediated by estrogen receptors (ERs), particularly ERbeta in the prostate epithelium. With the discovery of ERbeta isoforms, data from previous studies that focused principally on the wild-type ERbeta (ERbeta1) may not be adequate in explaining the still controversial role of ERbeta(s) in prostate carcinogenesis. In this study, using newly generated isoform-specific antibodies, immunohistochemistry (IHC) was performed on a tumor microarray comprised of 144 specimens. IHC results were correlated with pathological and clinical follow-up data to delineate the distinct roles of ERbeta1, ERbeta2, and ERbeta5 in PCa. ERbeta2 was commonly found in the cytoplasm and was the most abundant isoform followed by ERbeta1 localized predominantly in the nucleus, and ERbeta5 was primarily located in the cytoplasm. Logistic regression analyses demonstrated that nuclear ERbeta2 (nERbeta2) is an independent prognostic marker for prostate specific antigen (PSA) failure and postoperative metastasis (POM). In a Kaplan-Meier analysis, the combined expression of both nERbeta2 and cytoplasmic ERbeta5 identified a group of patients with the shortest POM-free survival. Cox proportional hazard models revealed that nERbeta2 predicted shorter time to POM. In concordance with IHC data, stable, ectopic expression of ERbeta2 or ERbeta5 enhanced PCa cell invasiveness but only PCa cells expressing ERbeta5 exhibited augmented cell migration. This is the first study to uncover a metastasis-promoting role of ERbeta2 and ERbeta5 in PCa, and show that the two isoforms, singularly and conjointly, have prognostic values for PCa progression. These findings may aid future clinical management of PCa.