Autism spectrum disorders (ASDs) include a range of developmental disorders that share a core of neurobehavioral deficits manifested by abnormalities in social interactions, deficits in communication, restricted interests, and repetitive behaviors. Several reports showed that mutations in different high-risk ASD genes, including SHANK3 and CNTNAP2, lead to ASD. However, to date, the underlying molecular mechanisms have not been deciphered, and no effective pharmacological treatment has been established for ASD. Recently, we reported a dramatic increase of nitric oxide (NO) in ASD mouse models. NO is a multifunctional neurotransmitter that plays a key role in different neurological disorders. However, its role in ASD has not yet been investigated. To reveal the novel molecular, cellular, and behavioral role of NO in ASD, we conducted multidisciplinary experiments using cellular and mouse models as well as clinical samples. First, we treated WT mice with an NO donor, which led to an autism-like phenotype. Next, we measured and found high levels of nitrosative stress biomarkers in both the Shank3 and Cntnap2 ASD mouse models. Treating both mouse models with a selective neuronal NO synthase (nNOS) inhibitor led to a reversal in the molecular, synaptic, and behavioral ASD phenotypes. Using a primary neuronal cell culture, we confirmed that NO is specifically involved in neurons in ASD pathology. Next, using genetic manipulations in the human SH-SY5Y cell line, we found that nNOS plays a key role in the pathology. Finally, we examined human plasma samples from 19 low-functioning ASD patients, compared to 20 typically developed volunteers, and found a significant elevation in the NO levels in the ASD patients. Furthermore, using the SNOTRAP technology, which is an innovative mass spectrometric method to identify the SNO-proteome (SNO: NO-mediated post-translational modification), we revealed that the complement systems in the synaptic and neuronal development processes are enriched in the ASD group. This work indicates, for the first time, that NO plays a pathological role in ASD development. Our findings will open future and novel directions to examine NO in diverse mutations on the autism spectrum as well as other neurodevelopmental disorders and psychiatric diseases. Most importantly, it suggests a novel treatment strategy for ASD.One sentence summary Nitric oxide plays a key role in ASD pathology development and progression, and targeting its production leads to a reversal in the autistic phenotype.Competing Interest StatementThe authors have declared no competing interest.