BACKGROUND: Androgen deprivation therapy (ADT) is the main clinical treatment for patients with advanced prostate cancer (PCa). However, PCa eventually progresses to castration-resistant prostate cancer (CRPC), largely because of androgen receptor variation and increased intratumoral androgen synthesis. Several studies have reported that one abnormal lipid accumulation is significantly related to the development of PCa. Melatonin (MLT) is a functionally pleiotropic indoleamine molecule and a key regulator of energy metabolism. The aim of our study is finding the links between CRPC and MLT and providing the basis for MLT treatment for CRPC. METHODS: We used animal CRPC models with a circadian rhythm disorder, and PCa cell lines to assess the role of melatonin in PCa. RESULTS: We demonstrated that MLT treatment inhibited tumor growth and reversed enzalutamide resistance in animal CRPC models with a circadian rhythm disorder. A systematic review and meta-analysis demonstrated that MLT is positively associated with an increased risk of developing advanced PCa. Restoration of carboxylesterase 1 (CES1) expression by MLT treatment significantly reduced lipid droplet (LD) accumulation, thereby inducing apoptosis by increasing endoplasmic reticulum stress, reducing de novo intratumoral androgen synthesis, repressing CRPC progression and reversing the resistance to new endocrine therapy. Mechanistic investigations demonstrated that MLT regulates the epigenetic modification of CES1. Ces1-knockout (Ces-/- ) mice verified the important role of endogenous Ces1 in PCa. CONCLUSIONS: Our findings provide novel preclinical and clinical information about the role of melatonin in advanced PCa and characterize the importance of enzalutamide combined with MLT administration as a therapy for advanced PCa.