Excitotoxicity has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). More recently, glial involvement has been shown to be essential for ALS-related motoneuronal death. Here, we identified an N-methyl-D-aspartate (NMDA) receptor co-agonist, D-serine (D-Ser), as a glia-derived enhancer of glutamate (Glu) toxicity to ALS motoneurons. Cell death assay indicated that primary spinal cord neurons from ALS mice were more vulnerable to NMDA toxicity than those from control mice, in a D-Ser-dependent manner. Levels of D-Ser and its producing enzyme, serine racemase, in spinal cords of ALS mice were progressively elevated, dominantly in glia, with disease progression. In vitro, expression of serine racemase was induced not only by an extracellular pro-inflammatory factor, but also by transiently expressed G93A-superoxide dismutase1 in microglial cells. Furthermore, increases of D-Ser levels were also observed in spinal cords of both familial and sporadic ALS patients. Collectively, Glu toxicity enhanced by D-Ser overproduced in glia is proposed as a novel mechanism underlying ALS motoneuronal death, and this mechanism may be regarded as a potential therapeutic target for ALS.