Excitatory neurotransmitter signaling through glutamate receptors modulates cognitive functions such as memory and learning, which are usually impaired in autism spectrum disorders (ASD). The aim of this study was to assess the clinical significance of plasma glutamate levels in ASD. Fifty-one children diagnosed with ASD, 51 typically developing children, and 51 children with intellectual disability matched for sex and age were assessed for plasma glutamate at admission. Plasma levels of glutamate were measured by liquid chromatography-tandem mass spectrometry and the severity of ASD was evaluated using the Childhood Autism Rating Scale Score. We found that the mean plasma glutamate levels were significantly (P<0.0001) higher in children with ASD compared with healthy controls and intellectual disability controls [36.1 (SD: 8.3) vs. 23.4 (4.2) vs. 24.7 (4.6) µM; P<0.001, respectively]. Levels of glutamate increased with increasing severity of ASD as defined by the Childhood Autism Rating Scale score. Receiver operating characteristics to diagnose ASD showed areas under the curve of glutamate of 0.92 [95% confidence interval (CI), 0.87-0.96], which was superior to high-sensitivity C-reactive protein [0.64 (95% CI, 0.55-0.75), P<0.001] and homocysteine (area under the curve, 0.72; 95% CI, 0.64-0.81; P<0.000). In multivariate logistic regression analysis, glutamate was an independent diagnosis indicator of ASD with an adjusted odds ratio of 1.362 (95% CI, 1.164-1.512; P<0.0001). The present study shows that autistic children had higher plasma levels of glutamate and elevated plasma glutamate levels may play an important role in the pathogenesis of autism. Further larger studies are required to support our findings.