Histone methylation is essential for gene expression control. Trimethylated lysine 27 of histone 3 (H3K27me3) is controlled by the balance between the activities of JMJD3 demethylase and EZH2 methyltransferase. This epigenetic mark has been shown to be deregulated in prostate cancer, and evidence shows H3K27me3 enrichment on gene promoters in prostate cancer. To study the impact of this enrichment, a transcriptomic analysis with TaqMan Low Density Array (TLDA) of several genes was studied on prostate biopsies divided into three clinical grades: normal (n = 23) and two tumor groups that differed in their aggressiveness (Gleason score ≤ 7 (n = 20) and >7 (n = 19)). ANOVA demonstrated that expression of the gene set was upregulated in tumors and correlated with Gleason score, thus discriminating between the three clinical groups. Six genes involved in key cellular processes stood out: JMJD3, EZH2, MGMT, TRA2A, U2AF1 and RPS6KA2. Chromatin immunoprecipitation demonstrated collocation of EZH2 and JMJD3 on gene promoters that was dependent on disease stage. Gene set expression was also evaluated on prostate cancer cell lines (DU 145, PC-3 and LNCaP) treated with an inhibitor of JMJD3 (GSK-J4) or EZH2 (DZNeP) to study their involvement in gene regulation. Results showed a difference in GSK-J4 sensitivity under PTEN status of cell lines and an opposite gene expression profile according to androgen status of cells. In summary, our data describe the impacts of JMJD3 and EZH2 on a new gene signature involved in prostate cancer that may help identify diagnostic and therapeutic targets in prostate cancer.