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 RORγ as a novel therapeutic target for CRPC. Here, we reveal that elaiophylin (Elai), an antibiotic from Actinomycete streptomyces, is a novel RORγ antagonist and showed potent antitumor activity against CRPC in vitro and in vivo. We demonstrated that Elai selectively binded to RORγ protein and potently blocked RORγ 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 RORγ to its genomic DNA response element (RORE), suppressed the expression of RORγ target genes AR and AR 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 RORγ inhibitor that can be used as a drug candidate for the treatment of human CRPC. © 2020 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences