AbstractProstate cancer (PCa) is the most commonly diagnosed cancer in men. Expanding evidence suggests a significant association between cancer progression and RNA modifications. However, our knowledge of the link between m5C and hm5C pathways with PCa is limited. Therefore, we aimed to explore the diagnostic and prognostic values of m5C and hm5C regulators in PCa. In this study, genetic alterations in m5C and hm5C regulators were identified using publicly available databases. Differentially expressed genes in these pathways between tumor and nontumor samples, correlation among m5C and hm5C pathway members, and prognostic value of the regulators were evaluated. Furthermore, enrichment of gene ontology (GO) terms and KEGG pathways was carried out. Obtained results unveiled the mRNA level differences as the key genetic alterations for m5C and hm5C regulators between tumor and nontumor samples. UHRF1, TET3, and NEIL1 were significantly upregulated in tumor samples compared to nontumor ones, whereas EGR1 was significantly downregulated. UHRF1, DNMT1, NSUN2, NSUN4, C1orf77, C3orf37, WDR77, NEIL1, and TDG genes were identified as candidate prognostic markers of overall survival. The upregulated genes in patient samples with genetic alterations in m5C and hm5C pathways enriched cell cycle-related processes. In summary, our findings suggest that the m5C and hm5C regulators might play a role in PRAD development by activation of proliferation, and the UHRF1, NSUN2, and NEIL1 genes have the potential to be utilized as clinical biomarkers.