BACKGROUND: Castration-resistant prostate cancer (CRPC) has been a major cause of tumor-associated death among men worldwide. The discovery of novel therapeutic medicines for CRPC remains imperative. Atractylenolide I (ATR-I), a prominent bioactive component from Atractylodes macrocephala, exhibits powerful anticancer potentials in various malignancies. Nevertheless, the ATR-I's activity on CRPC has not been reported. METHODS: An enzalutamide-resistant (EnzR) cell line was successfully constructed. CCK-8, EdU, wound healing, Transwell assays, flow cytometry, and xenograft tumor models were applied to investigate the antitumor activity of ATR-I against CRPC. The changes in the gene expression profiles after ATR-I treatment were analyzed using RNA sequencing. RESULTS: ATR-I suppressed the proliferative and migratory abilities of AR CONCLUSIONS: These findings identified ATR-I as a promising therapeutic drug for overcoming enzalutamide resistance in CRPC patients and increased our understanding about its antitumor mechanisms.