Silibinin, a diastereoisomeric mixture extracted from Silybum marianum L, with established anti-prostate cancer activity, has been associated with considerable anti-neoplastic ability, in a variety of human cancer types, through interference with the epigenetic machinery. In prostate carcinoma (PCa), high expression of polycomb repressive complex 1 (PRC1) and 2 (PRC2) members, that belong to polycomb group (PcG) proteins, is associated with transcriptional silencing of tumor suppressor genes through histone modifications and chromatin condensation. Our previous results revealed that silibinin reduced the expression levels of PRC2 complex members (EZH2, EED, SUZ12), an ability accompanied by increased H3K27me3 marks. In the current report, treatment of DU145 and PC3 prostate cancer cells with clinically-achievable concentrations (25-75μg/mL) of silibinin, resulted in reduced protein expression levels of PRC1 complex members (RING1a, RING1b and BMI1), in a dose-dependent manner. Next, human epigenetic chromatin modification enzymes-focused DNA microarray and real-time quantitative reverse transcription-PCR (qRT-PCR) analyses, revealed that silibinin modulated differentially the expression of important enzymes, related with the pathophysiology of the disease, at the epigenetic level. Specifically, significant alterations were observed in the expression profile of enzymes associated with gene expression regulation through modification of chromatin configuration, including family members of: i) histone methyltransferases, ii) histone acetyl-transferases,iii) histone demethylases and iv) histone deacetylases along with enzymes inducing gene silencing (via DNA methylation) and regulation of cell cycle progression. Our results suggest that the anticancer activity of silibinin is partially mediated by the disruption of central processes in chromatin configuration-remodeling and alteration of enzymes of the epigenomic landscape that regulate prostate cancer progression.