Enoyl-CoA hydratase is an enzyme that hydrates the double bond between the second and third carbons on acyl-CoA. The enzyme catalyzes the second step in the break down of fatty acids or the second step of β-oxidation in fatty acid metabolism. Here, we report the crystal structure of enoyl-CoA hydratase from Bacillus anthracis str. 'Ames Ancestor' determined at 1.82 A resolution. The crystal structure shows a hexamer formation. The hexamer is a dimer of trimers. Each monomer is folded into a right-handed spiral of four turns, followed by two small domains, which are involved in trimerization. Each turn of the spiral consists of two beta-strands and an alpha-helix. The mechanism for the hydratase/dehydratase reaction follows a syn-stereochemistry, a preference that is opposite to the nonenzymatic reaction. The active-site architecture agrees with this stereochemistry.