Δ 3 -Δ 2 -Enoyl-CoA isomerase (EC 5.3.3.8) is a key enzyme for the β-oxidation of unsaturated fatty acids. The cDNA of the full-length rat liver Δ 3 -Δ 2 -enoyl-CoA isomerase was previously cloned as pAG847. PCR methodologies were used to subclone the gene encoding the functional Δ 3 -Δ 2 -enoyl-CoA isomerase from pAG847 with primers that were designed to add six continuous histidine codon to the 5 ' primer. The PCR product was inserted into a pLM1 expression vector and overexpressed in Escherichia coli. The soluble expressed protein was purified with a nickel HiTrap chelating metal affinity column to apparent homogeneity based on Coomassie blue-stained SDS-PAGE and the molecular weight of the protein subunit was 30kDa. The purified protein had a dimeric structure composed of identical subunits, and the molecular weight of the enzyme determined by gel chromatography was 60kDa. Kinetic studies have been carried out and K M of 81μM and V m a x of 292μmol/min/mg were determined. The specific activity of the protein is 201U/mg, which is significantly higher than that reported before for the same protein isolated from a natural source. The one-step purification of the highly active Δ 3 -Δ 2 -enoyl-CoA isomerase will greatly facilitate the further investigation of this enzyme through site-directed mutagenesis and enzyme catalyzed reactions with substrate analogues.