Prostaglandin (PG) D₂ is the most abundant prostanoid produced in the central nervous system of mammals and has been implicated in the modulation of neural functions such as sleep induction, nociception, regulation of body temperature, and odor responses. We generated gene-knockout mice for lipocalin-type PGD₂ synthase (L-PGDS) and found that the intrathecal administration of PGE₂, an endogenous pain-producing substance, failed to elicit allodynia (touch-evoked pain), which is one typical phenomenon of neuropathic pain, whereas it evoked thermal hyperalgesia, in L-PGDS−/− mice. We also found that the allodynic response induced by the γ-aminobutyric acid (GABA)_A receptor antagonist bicuculline was selectively abolished in the L-PGDS−/− mice, among excitatory and inhibitory agents that induced allodynia in wild-type mice. Interestingly, simultaneous injection of a femtogram amount of PGD₂ with PGE₂ or bicuculline induced allodynia in L-PGDS−/− mice to the same extent as in wild-type mice. The PGE₂- or bicuculline-evoked allodynia in wild-type and in PGD₂-supplemented L-PGDS−/− mice was blocked by a PGD₂ receptor antagonist given in a femtogram amount. These results reveal that endogenous PGD₂ is essential for both PGE₂- and bicuculline-induced allodynia.