A bioenergetic theory of prostate malignancy proposed that normal citrate-producing prostate epithelial cell become citrate-oxidizing cells, in which mitochondrial aconitase (mACON) is not limiting, providing the energy required for the onset and progression of malignancy and metastasis. However, no direct evidence has been approved to support the hypothesis. A full-length cDNA encoding human skeletal muscle mACON cDNA was cloned and sequenced. mACON cDNA contains 19-bp 5' untranslated region, a 2343-bp coding segment, and 376-bp 3' untranslated region. This precursor enzyme contains mitochondrial targeting sequence of 27 amino acid residues and mature enzyme of 753 amino acids residues. A human anti-mACON overexpression vector containing the 1171-bp mACON cDNA fragment in the reverse orientation was stable transfected into human prostate carcinoma cells, PC-3 and DU145 cells. Results showed that mACON antisense blocked 40-60% mACON expression and enzymatic activity which induced decrease in the intracellular ATP biosynthesis but increase citrate secretion in the human prostate carcinoma cells. mACON antisense-transfected cells have lower cell proliferation ratio than the mock of DNA-transfected cells. Our study demonstrated the key role of the mACON in the cellular bioenergy and cell proliferation of human prostate carcinoma cells.