O-Linked N-acetylglucosamine, or O-GlcNAc, is a dynamic post-translational modification that cycles on and off serine and threonine residues of nucleocytoplasmic and mitochondrial proteins. In addition to cancer and inflammation diseases, O-GlcNAc modification appears to play a critical role during cell apoptosis and stress response, although the precise mechanisms are still not very clear. Here we found that nitric oxide synthase adaptor (NOS1AP), which plays an important part in glutamate-induced neuronal apoptosis, carries the modification of O-GlcNAc. Mass spectrometry analysis identified Ser47, Ser183, Ser204, Ser269, Ser271 as O-GlcNAc sites. Higher O-GlcNAc of NOS1AP was detected during glutamate-induced neuronal apoptosis. Furthermore, with O-GlcNAc sites of NOS1AP mutated, the interaction of NOS1AP and neuronal nitric oxide syntheses (nNOS) decreases. Finally, during glutamate-induced neuronal apoptosis, decreasing the O-GlcNAc modification of NOS1AP results in more severe neuronal apoptosis. All these results suggest that O-GlcNAc modification of NOS1AP exerts protective effects during glutamate-induced neuronal apoptosis.