Due to heterogeneous multifocal nature of prostate cancer (PCa), there is currently a lack of biomarkers that stably distinguish it from benign prostatic hyperplasia (BPH), predict clinical outcome and guide the choice of optimal treatment. In this study, RNA-seq analysis was applied to formalin-fixed paraffin-embedded (FFPE) tumor and matched normal tissue samples collected from Russian patients with PCa and BPH. We identified 3384 genes differentially expressed (DE) (FDR < 0.05) between tumor tissue of PCa patients and adjacent normal tissue as well as both tissue types from BPH patients. Overexpression of four of the genes previously not associated with PCa (ANKRD34B, NEK5, KCNG3, and PTPRT) was validated by RT-qPCR. Furthermore, the enrichment analysis of overrepresented microRNA and transcription factor (TF) recognition sites within DE genes revealed common regulatory elements of which 13 microRNAs and 53 TFs were thus linked to PCa for the first time. Moreover, 8 of these TFs (FOXJ2, GATA6, NFE2L1, NFIL3, PRRX2, TEF, EBF2 and ZBTB18) were found to be differentially expressed in this study, making them not only candidate biomarkers of prostate cancer but also potential therapeutic targets. Overall design: Whole transcriptome profiling of tumor tissue and matched adjacent normal tissue from 15 patients with PCa and 2 with BPH.