Mitochondrial dynamics regulated by mitochondrial fusion and fission maintain mitochondrial functions, whose alterations underline various human diseases. The crosstalk between cellular metabolites and mitochondrial dynamics under energy stress remains obscure. We found that inositol is a critical metabolite directly restricting AMPK-dependent mitochondrial fission independently of its classical mode as a precursor for phosphoinositide generation. Metabolic stress or mitochondrial damage causes inositol decline in cells and mice to elicit AMPK-dependent mitochondrial fission. Mechanistically, inositol directly binds to AMPKg and competes with AMP for AMPKg binding, leading to restriction of AMPK activation and mitochondrial fission. Our study suggests that the AMP/inositol ratio is a critical determinant for AMPK activation and establishes a model in which AMPK activation requires inositol decline to release AMPKg for AMP binding. In terms of prostate cancer, acquisition of prostate cancer stem cells (PCSCs) manifested during androgen ablation therapy (ABT) contributes to castration-resistant prostate cancer (CRPC). However, little is known about the specific metabolites critically orchestrating this process. We also found IMPA1-derived inositol enriched in PCSCs is a key metabolite crucially maintaining PCSCs for CRPC progression and ABT resistance. Notably, conditional Impa1 knockout in prostate abrogates the pool and properties of PCSCs leading to CRPC progression and prolonging mouse survival in TRAMP mouse model. IMPA1/inositol is upregulated in human prostate cancer and its overexpression predicts poor survival outcome. Genetically and pharmacologically targeting IMPA1/inositol abrogates CRPC and overcomes ABT resistance in various CRPC xenografts and patient-derived xenograft (PDX) tumor models. Our study identifies inositol represents a key metabolite to maintain the homeostasis of mitochondrial dynamics and PCSCs for CRPC and ABT resistance.