Currently, no clinical methods reliably predict the development of castration-resistant prostate cancer (CRPC) that occurs almost universally in men undergoing androgen deprivation therapy. Hyperpolarized (HP) 13C magnetic resonance imaging (MRI) could potentially detect the incipient emergence of CRPC based on early metabolic changes. To characterize metabolic shifts occurring upon the transition from androgen-dependent to castration-resistant prostate cancer (PCa), the metabolism of [U-13C]glucose and [U-13C]glutamine was analyzed by nuclear magnetic resonance spectroscopy. Comparison of steady-state metabolite concentrations and fractional enrichment in androgen-dependent LNCaP cells and transgenic adenocarcinoma of the murine prostate (TRAMP) murine tumors versus castration-resistant PC-3 cells and treatment-driven CRPC TRAMP tumors demonstrated that CRPC was associated with upregulation of glycolysis, tricarboxylic acid metabolism of pyruvate; and glutamine, glutaminolysis, and glutathione synthesis. These findings were supported by 13C isotopomer modeling showing increased flux through pyruvate dehydrogenase (PDH) and anaplerosis; enzymatic assays showing increased lactate dehydrogenase, PDH and glutaminase activity; and oxygen consumption measurements demonstrating increased dependence on anaplerotic fuel sources for mitochondrial respiration in CRPC. Consistent with ex vivo metabolomic studies, HP [1-13C]pyruvate distinguished androgen-dependent PCa from CRPC in cell and tumor models based on significantly increased HP [1-13C]lactate.