Summary Prostate cancer is the second leading cause of cancer deaths in the United States. While androgen ablation therapy (ABT) is the mainstay of therapy for men with prostate cancer, most patients with prostate cancer will inevitably develop castration-resistant prostate cancer (CRPC), which no longer responds to ABT treatment. Thus, understanding of the mechanisms leading to CRPC and ABT resistance is urgently needed. Several mechanisms account for the occurrence of CRPC, such as activation of 3-kinase/Akt signaling pathway, which plays a critical role in cell proliferation and cell survival, and higher expression of AR and AR splicing variants, which facilitate prostate cancer cell survival under very low androgen concentrations. Of note is that prostate cancer stem cells (PCSCs) also known as cancer initiating cells (CICs), which account for a small cell population prostate cancer cells, are critically involved in the development of CRPC. But how PCSCs are regulated and how they can be pharmacologically targeted are currently not well understood. While several stem cell factors such as SOX2 playing an important role in maintaining PCSC pool and functions are identified, there is no effective strategy to block the action of these proteins in order to eradicate PCSCs within the cancer. Thus, identifying key druggable targets maintaining PCSCs could provide novel paradigms and effective strategies for prostate cancer therapy and overcoming ABT resistance. Our goal in this study is to identify a novel mechanism underlying PCSC maintenance, which could be harnessed to develop an effective strategy for CRPC targeting. Using systematic metabolomics and transcriptomics in conjunction with biochemical validation and in vivo tumor development assays, we unraveled a novel oncogenic and metabolic signaling pathway, which was elevated during ABT treatment and in advanced human prostate cancer, is critical for maintaining the stemness and pool of PCSCs and CRPC development. Of note, we observed aberrant AMPK activation and AMPK-dependent mitochondria fission upon loss of this metabolic signaling, correlated with the defect in stemness and pool of PCSCs and prostate cancer progression. In light of these findings, we hypothesized that this newly discovered metabolic signaling is crucial for restricting aberrant AMPK-dependent mitochondrial fission to maintain the pool and stemness of PCSCs, thereby leading to CRPC and ABT resistance. Our hypothesis has been formulated based on our solid and innovative preliminary results. In this proposal, we proposed three specific aims to test this paradigm-shift hypothesis. Our study has not only revolutionized and significantly advanced our understanding of cancer metabolism in PCSC regulation, but also offered a promising strategy to treat advanced prostate cancer and overcoming ABT resistance.