Although estrogens have been long implicated in the prostate carcinogenesis, direct evidence showing their carcinogenicity on prostatic epithelial cells has not yet been clearly demonstrated. In this study, we treated an immortalized, non-transformed and androgen-responsive rat prostatic epithelial cell line NRP-152 with 17β-estradiol (E2) at concentrations 1–3μM for period 2–6weeks. After in vitro treatment, we evaluated the anchorage-independent growth of E2-treated NRP-152 cells by soft agar assay and isolated the colonies formed by the transformed E2-NRP-152 cells in soft agar for further growth phenotype characterization. Our results showed that the isolated E2-NRP-152 clones displayed neoplastic transformation phenotype, as demonstrated by their capacity of forming colonies in soft agar and tumors in immunodeficient nude mice, while losing their spheroid formation capacity in Matrigel 3D-culture. Western blot and RT-PCR analyses showed that the transformed E2-NRP-152 cells expressed increased levels of ERα and several putative prostate cancer stem cell markers (integrins α2β1, CD44, CD133, ABCG2 and CXCR4) but decreased levels of ERβ and AR. Comet assay revealed that E2-treatment also induced formation of comet cells, indicating that E2 caused DNA damage to the NRP-152 cells. Our present findings demonstrated that in vitro E2 exposure could neoplastically transform the rat prostatic epithelial cells, indicating that E2 is carcinogenic to the prostatic epithelial cells.