Prostate cancer (PCa) patients who progress to metastatic castration-resistant PCa (mCRPC) mostly have poor outcomes due to the lack of effective therapies. Our recent study established the orphan nuclear receptor ROR as a novel therapeutic target for CRPC. Here, we reveal that elaiophylin (Elai), an antibiotic from , is a novel ROR antagonist and showed potent antitumor activity against CRPC and . We demonstrated that Elai selectively binded to ROR protein and potently blocked ROR transcriptional regulation activities. Structure-activity relationship studies showed that Elai occupied the binding pocket with several key interactions. Furthermore, Elai markedly reduced the recruitment of ROR to its genomic DNA response element (RORE), suppressed the expression of ROR target genes and variants, and significantly inhibited PCa cell growth. Importantly, Elai strongly suppressed tumor growth in both cell line based and patient-derived PCa xenograft models. Taken together, these results suggest that Elai is novel therapeutic ROR inhibitor that can be used as a drug candidate for the treatment of human CRPC.