Prolactin (PRL) is a pituitary polypeptide hormone characterized by multiple biological actions including stimulation of growth in the prostate, breast and ovarian epithelial cells. A sizable body of reports has presented evidences to indicate the involvement of PRL in the pathogenic process of cancers of the reproductive system, such as prostate and breast cancers. PRL exerts its effects by dimerizing its receptor (PRLR) on the plasma membrane, and initiating cellular Jak-Stat signal pathway. We have previously cloned from prostate cancer cells a natural variant of PRLR in which the S2 subdomain of the extracellular domain is missing (ΔS2). Our preliminary data showed that ΔS2 PRLR was able to dimerize and to constitutively activate the β-casein promoter (in the absence of its ligand, PRL) in breast and prostate epithelial cells. Enhancer of zeste homologue 2 (EZH2), an important histone-modifying enzyme, is able to trimethylate histone 3 on lysine 27 (H3K27Me3), consequently leading to gene silencing, especially silencing of tumor suppressor genes such as p53. We hypothesized that ΔS2 PRLR played an important pathogenic role in prostate cancer through, at least partly, alterations in the expression of EZH2 and the trimethylation of histone 3 on lysine 27. In the present study, overexpression of ΔS2 PRLR in prostate epithelial cells was achieved by infection with an adenoviral vector carrying the cDNA. The viable cell number overexpressing ΔS2 PRLR was assessed using MTS reagent. Western blot, chromatin immunoprecipitation (ChIP) assay and acid histone extraction were applied to detect expression of EZH2 as well as trimethylation of histone 3, respectively. In prostate epithelial cells, overexpression of ΔS2 PRLR increased the levels of EZH2 methyltransferase mRNA and protein, induced EZH2 methyltransferase recruitment to chromatin, increased the trimethylation of histone 3 on lysine 27, and decreased expression of the p53 gene. We concluded that ΔS2 PRLR plays an important pathogenic role in prostate cancer through epigenetic covalent modification leading to chromatin remodeling. Hypertrimethylation on H3K27 of the p53 gene promoter region due to elevated expression of ΔS2 PRLR by alternative splicing of the pre-mRNA in its full-length form might serve as a new mechanism underlying human prostate cancer.