OBJECTIVE: De novo lipogenesis (DNL) is associated with prostate cancer (PCa) progression, while fatty acid synthase (FASN) overexpression is a hallmark of DNL. Palmitate, its main product, is a saturated fatty acid that supports PCa growth. Polyunsaturated fatty acids (PUFAs), which can be acquired from the microenvironment, undergo peroxidation more readily and affect membrane fluidity. Docosahexaenoic acid (DHA) is a prototype PUFA omega-3 produced inefficiently in human cells. Its levels are low in PCa cells compared to normal cells. We hypothesize that excess DHA may reprogram lipid metabolism and induce cell growth suppression. METHODS: Androgen-responsive LNCaP, castration-resistant cells C4-2 and 22Rv1, human PCa castration-resistant organoids, and prostate cancer xenografts were exposed to DHA. RESULTS: DHA accumulated into lipid droplets as triacylglycerols and cholesterol esters, led to increased phospholipid acyl chain unsaturation and altered phospholipid ratio, a known trigger of endoplasmic reticulum (ER) stress. DHA caused a decrease in sterol regulatory element-binding protein (SREBP) transcriptional program, which, in turn, led to decreased expression of FASN. The subsequent reduction in DNL caused downregulation of the androgen receptor (AR) and its splice variant AR-V7. In addition, β-oxidation was enhanced, and DHA was preferentially oxidized over palmitate. Glucose oxidation also increased in the presence of DHA. Finally, DHA led to ROS overproduction, oxidative damage, and ER stress. CONCLUSIONS: DHA reduces the growth of hormone-sensitive and castration-resistant PCa both in vitro and in vivo via deregulation of lipid metabolism.