ObjectivesDue to the heterogeneity of PCa, the clinical indicators used for PCa can't satisfy risk prognostication and personalized treatment. It is imperative to develop novel biomarkers for prognosis prediction and therapy response in PCa. Accumulating evidence shows that non-mutational epigenetic reprogramming, independent from genomic instability and mutation, serves as a newly added hallmark in cancer progression.MethodsIn this study, we integrated multi-center cohorts (N > 1300) to develop a RNA 5-methylcytosine regulator-based signature, the m5C score. We performed unsupervised clustering and LASSO regression to identify novel m5C-related subtypes and calculate the m5C score. Then we assessed the role of m5C cluster and m5C score in several clinical aspects such as prognosis in various molecular subtypes, responses to chemotherapy, androgen receptor signaling inhibitor (ARSI) therapy and immunotherapy in PCa. Finally, we validated the cancer-promoting performance of ALYREF through clinical data analysis and experiments in vivo and in vitro.ResultsThe investigation revealed that the m5C score could accurately predict the biochemical recurrence (BCR) in different subtypes (the PAM50 subtypes and immunophenotypes) and the responses to chemotherapy, ARSI therapy, and immunotherapy (PD1/PD-L1). A high m5C score indicated a poor BCR prognosis in every subtype of PCa, unfavorable responses in ARSI therapy and immunotherapy (PD1/PD-L1). Moreover, the m5C reader gene termedALYREF,yielding the highest weighed coefficient, promoted PCa progression through in silico analysis and experimental validations (in vivo and in vitro).ConclusionsThe m5C signature can function in many aspects of PCa, such as the development and prognosis of the disease, and multiple therapy responses. Further, the m5C reader, ALYREF, was identified as a prognostic biomarker and a potential therapeutic target for PCa. The m5C signature could act as a brand-new tool for predicting the prognosis of patients in different molecular subtypes and patients’ therapy responses and promoting customized treatments.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12672-023-00671-w.