Hypoxia induces metabolic alteration in cancer cells by stabilizing hypoxia-inducible factor 1α (HIF-1α (HIF1A)), which regulates the bioenergetic genes of glycolysis and lipid metabolic pathways. However, the target genes of hypoxia-induced metabolic alterations in the prostate remain uncertain. Mitochondrial aconitase (mACON) (ACONM) is an enzyme that is central to carbohydrate and energy metabolism and is responsible for the interconversion of citrate to isocitrate as part of the citric acid cycle in the human prostate. We evaluated the effects of the molecular mechanisms of hypoxia onmACONgene expression in PC-3 and LNCaP human prostate carcinoma cells. Immunoblotting assays revealed that hypoxia modulated mACON and lactate dehydrogenase A (LDHA) protein expression, while these effects were attenuated whenHIF-1αwas knocked down. Hypoxia induced fatty acid synthase (FASN) in PC-3 cells while hypoxia blockedFASNgene expression in LNCaP cells after 24-h incubation. Results of real-time RT-qPCR, immunoblotting, and transient gene expression assays revealed that hypoxia treatment or co-transfection withHIF-1αexpression vector enhanced gene expression ofmACON, implying that hypoxia modulatedmACONat the transcriptional level. Hypoxia-inducedmACONpromoter activity is dependent on the DNA fragment located at −1013 to −842 upstream of the translation initiation site.l-mimosine, an iron chelator, stabilized HIF-1α but downregulatedmACONgene expression, suggesting that iron chelation blocked the hypoxia-inducedmACONgene expression. These results suggest that hypoxia dysregulates the expressions ofLDHA,FASN, andmACONgenes, and the hypoxia-inducedmACONgene expression is via the HIF-1α-dependent and iron-dependent pathways in prostate carcinoma cells.