Human prostaglandin D synthase (L-PGDS) is a lipocalin-type enzyme involved in the metabolism of arachidonic acid and plays a key role in the regulation of sleep, allergy, pain sensation, and the development of male reproductive organs. Here, using a combination of crystallographic, biochemical, mutagenesis, and kinetic studies, we have gained insights into the mode of ligand binding by human L-PGDS and have identified residues involved in catalysis. Interestingly, structural evidence reveals that 2 molecules of fatty acids, one molecule each of oleic and palmitoleic acid, bind inside the β barrel. The oleic acid is buried and binds in a highly basic patch in proximity to the catalytically critical Cys65, mimicking the binding of prostaglandin H(2). The palmitoleic acid sits in a relatively neutral region with very few interactions with the protein. Mutating Met64, Leu79, Phe83, or Leu131 to alanine reduced the catalytic efficiency by almost 10-fold, while K59A and Y149A mutations enhanced the catalytic efficiency by >2-fold. Met64 seems to function as a kinetic clamp, pushing the thiol group of Cys65 close to the site of nucleophilic attack during catalysis.