Abstract The hydratase/isomerase superfamily consists of enzymes having a common evolutionary origin but acting in a wide variety of metabolic pathways. Many of the superfamily members take part in β-oxidation, one of the processes of fatty acid degradation. One of these β-oxidation enzymes is the Δ3-Δ 2-enoyl-CoA isomerase, which is required for the metabolism of unsaturated fatty acids. It catalyzes the shift of a double bond from the position C3 of the substrate to the C2 position. In this study, the Δ 3-Δ 2-enoyl-CoA isomerase from the yeast Saccharomyces cerevisiae was identified, overexpressed as a recombinant protein and characterized. Subsequently, its structure and function were studied by X-ray crystallography. The yeast Δ 3-Δ 2-enoyl-CoA isomerase polypeptide contains 280 amino acid residues, which corresponds to a subunit size of 32 kDa. Six enoyl-CoA isomerase subunits assemble to form a homohexamer. According to structural studies, the hexameric assembly can be described as a dimer of trimers. The yeast Δ 3-Δ 2-enoyl-CoA isomerase is located in peroxisomes, the site of fungal β-oxidation, and is a necessary prerequisite for the β-oxidation of unsaturated fatty acids; the enoyl-CoA isomerase knock-out was unable to grow on such carbon sources. In the crystal structure of the yeast Δ 3-Δ 2-enoyl-CoA isomerase, two domains can be recognized, the N-terminal spiral core domain for catalysis and the C-terminal α-helical trimerization domain. This overall fold resembles the other known structures in the hydratase/isomerase superfamily. Site-directed mutagenesis suggested that Glu158 could be involved in the enzymatic reaction. Structural studies confirmed this, as Glu158 is optimally positioned at the active site for interaction with the substrate molecule. The oxyanion hole stabilizing the transition state of the enzymatic reaction is formed by the main chain NH groups of Ala70 and Leu126. The yeast Δ 3-Δ 2-enoyl-CoA isomerase hexamer forms by dimerization of two trimers, as in the other superfamily members. An extensive comparison of the five known structures of this family showed that the mode of assembly into hexamers is not a conserved feature of this superfamily, since the distance between the trimers and the orientation of the trimers with respect to each other varied. Marked differences were also detected between the two yeast enoyl-CoA isomerase crystal forms used in this study, one being crystallized at low pH and the other at neutral pH. The results suggest that the yeast Δ 3-Δ 2-enoyl-CoA isomerase could occur as a trimer at low pH.