Radiotherapy is a curative arsenal for prostate cancer (PCa), but radioresistance seriously compromises its effectiveness. Dysregulated RNA splicing factors are extensively involved in tumor progression. Nonetheless, the role of splicing factors in radioresistance remains largely unexplored in PCa. Here, 23 splicing factors that are differentially expressed between PCa and adjacent normal tissues across multiple public PCa databases are identified. Among those genes, polypyrimidine tract binding protein 1 (PTBP1) is significantly upregulated in PCa and is positively associated with advanced clinicopathological features and poor prognosis. Gain- and loss-of-function experiments demonstrate that PTBP1 markedly reinforces genomic DNA stability to desensitize PCa cells to irradiation in vitro and in vivo. Mechanistically, PTBP1 interacts with the heterogeneous nuclear ribonucleoproteins (hnRNP) associated with lethal yellow protein homolog (RALY) and regulates exon 5 splicing of DNA methyltransferase 3b (DNMT3B) from DNMT3B-S to DNMT3B-L. Furthermore, upregulation of DNMT3B-L induces promoter methylation of dual-specificity phosphatase-2 (DUSP2) and subsequently inhibits DUSP2 expression, thereby increasing radioresistance in PCa. The findings highlight the role of splicing factors in inducing aberrant splicing events in response to radiotherapy and the potential role of PTBP1 and DNMT3B-L in reversing radioresistance in PCa.