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D-Amino acid oxidase (DAAO) is a flavin adenine dinucleotide-dependent peroxisomal flavoenzyme which is almost exclusively expressed within astrocytes in the spinal cord. DAAO catalyzes oxidation of D-amino acids to hydrogen peroxide, which is a stable and less active reactive oxygen species, and may represent a final form of reactive oxygen species. This study tested the hypothesis that the spinal astroglial DAAO–hydrogen peroxide pathway plays an important role in the development of morphine antinociceptive tolerance.Rat and mouse formalin, hot-plate, and tail-flick tests were used, and spinal DAAO expression and hydrogen peroxide level were measured. Sample size of animals was six in each study group.Subcutaneous and intrathecal DAAO inhibitors, including 5-chloro-benzo[d]isoxazol-3-ol, AS057278, and sodium benzoate, completely prevented and reversed morphine antinociceptive tolerance in the formalin, hot-plate, and tail-immersion tests, with a positive correlation to their DAAO inhibitory activities. Intrathecal gene silencers, small interfering RNA/DAAO and small hairpin RNA/DAAO, almost completely prevented morphine tolerance. Intrathecal 5-chloro-benzo[d]isoxazol-3-ol and small interfering RNA/DAAO completely prevented increased spinal hydrogen peroxide levels after chronic morphine treatment. Intrathecal nonselective hydrogen peroxide scavenger phenyl-tert-N-butyl nitrone and the specific hydrogen peroxide catalyst catalase also abolished established morphine tolerance. Spinal dorsal horn astrocytes specifically expressed DAAO was significantly up-regulated, accompanying astrocyte hypertrophy after chronic morphine treatment.For the first time, the authors’ result identify a novel spinal astroglial DAAO–hydrogen peroxide pathway that is critically involved in the initiation and maintenance of morphine antinociceptive tolerance, and suggest that this pathway is of potential utility for the management of morphine tolerance and chronic pain.