We determined the crystal structure of human hematopoietic prostaglandin (PG) D synthase (H-PGDS) as the quaternary complex with glutathione (GSH), Mg²⁺, and an inhibitor, HQL-79, having anti-inflammatory activities in vivo, at a 1.45 Å resolution. In the quaternary complex, HQL-79 was found to reside within the catalytic cleft between Trp104 and GSH. HQL-79 was stabilized by interaction of a phenyl ring of its diphenyl group with Trp104 and by its piperidine group with GSH and Arg14 through water molecules, which form a network with hydrogen bonding and salt bridges linked to Mg²⁺. HQL-79 inhibited human H-PGDS competitively against the substrate PGH₂ and non-competitively against GSH with Ki of 5 and 3 M, respectively. Surface plasmon resonance analysis revealed that HQL-79 bound to H-PGDS with an affinity that was 12-fold higher in the presence of GSH and Mg²⁺ (Kd = 0.8 M) than in their absence. Mutational studies revealed that Arg14 was important for the Mg²⁺-mediated increase in the binding affinity of H-PGDS for HQL-79, and that Trp104, Lys112, and Lys198 were important for maintaining the HQL-binding pocket. HQL-79 selectively inhibited PGD₂ production by H-PGDS-expressing human megakaryocytes and rat mastocytoma cells with an IC50 value of about 100 M but only marginally affected the production of other prostanoids, suggesting the tight functional engagement between H-PGDS and cyclooxygenase. Orally administered HQL-79 (30 mg/kg body weight) inhibited antigen-induced production of PGD₂, without affecting the production of PGE₂ and PGF_2{alpha}, and ameliorated airway inflammation in wild-type and human H-PGDS-overexpressing mice. Knowledge about this structure of quaternary complex is useful for understanding the inhibitory mechanism of HQL-79 and should accelerate the structure-based development of novel anti-inflammatory drugs that inhibit PGD₂production specifically.