Alternol　is　a　natural　compound　isolated　from　fermentation　products　of　a　mutant　fungus.　Our　previous　studies　demonstrated　that　Alternol　specifically　kills　cancer　cells　but　spares　benign　cells.To　investigate　the　mechanism　underlying　alternol-induced　cancer　cell-specific　killing　effect,　we　took　a　comprehensive　strategy　to　identify　Alternol＇s　protein　targets　in　prostate　cancer　cells,　including　PC-3,　C4-2,　and　22RV1,　plus　benign　BPH1　cell　lines.　Major　experimental　techniques　included　biotin-streptavidin　pulldown　assay　coupled　with　mass-spectrometry,　in　vitro　enzyme　activity　assay　for　Krebs　cycle　enzymes　and　gas　chromatography-mass　spectrometry　(GC-MS)　for　metabolomic　analysis.Among　14　verified　protein　targets,　four　were　Krebs　cycle　enzymes,　fumarate　hydratase　(FH),　malate　dehydrogenase-2　(MDH2),　dihydrolipoamide　acetyltransferase　(DLAT)　in　pyruvate　dehydrogenase　complex　(PDHC)　and　dihydrolipoamide　S-succinyltransferase　(DLST)　in　a-ketoglutarate　dehydrogenase　complex　(KGDHC).　Functional　assays　revealed　that　PDHC　and　KGDHC　activities　at　the　basal　level　were　significantly　higher　in　prostate　cancer　cells　compared　to　benign　prostate　BPH1　cells,　while　alternol　treatment　reduced　their　activities　in　cancer　cells　close　to　the　levels　in　BPH1　cells.　Although　FH　and　MDH2　activities　were　comparable　among　prostate　cancer　and　benign　cell　lines　at　the　basal　level,　Alternol　treatment　largely　increased　their　activities　in　cancer　cells.　Metabolomic　analysis　revealed　that　Alternol　treatment　remarkably　reduced　the　levels　of　malic　acid,　fumaric　acid,　and　isocitric　acid　and　mitochondrial　respiration　in　prostate　cancer　cells.　Alternol　also　drastically　reduced　mitochondrial　respiration　and　ATP　production　in　PC-3　cells　in　vitro　or　in　xenograft　tissues　but　not　in　BPH1　cells　or　host　liver　tissues.Alternol　interacts　with　multiple　Krebs　cycle　enzymes,　resulting　in　reduced　mitochondrial　respiration　and　ATP　production　in　prostate　cancer　cells　and　xenograft　tissues,　providing　a　novel　therapeutic　strategy　for　prostate　cancer　treatment.