O-linked conjugation of ß-N-acetyl-glucosamine (O-GlcNAc) to serine and threonine residues is a post-translational modification process that senses nutrient availability and cellular stress and regulates diverse biological processes that are involved in neurodegenerative diseases and provide potential targets for therapeutics development. However, very little is known of the networks involved in the brain that are responsive to changes in the O-GlcNAc proteome. Pharmacological increase of protein O-GlcNAcylation by Thiamet G (TG) has been shown to decrease tau phosphorylation and neurotoxicity, and proposed as a therapy in Alzheimer's disease (AD). However, acute TG exposure impairs learning and memory, and protein O-GlcNAcylation is increased in the aging rat brain and in Parkinson's disease (PD) brains. To define the cortical O-GlcNAc proteome that responds to TG, we injected young adult mice with either saline or TG and performed mass spectrometry analysis for detection of O-GlcNAcylated peptides. This approach identified 506 unique peptides corresponding to 278 proteins that are O-GlcNAcylated. Of the 506 unique peptides, 85 peptides are elevated by > 1.5 fold in O-GlcNAcylation levels in response to TG. Using pathway analyses, we found TG-dependent enrichment of O-GlcNAcylated synaptic proteins, trafficking, Notch/Wnt signaling, HDAC signaling, and circadian clock proteins. Significant changes in the O-GlcNAcylation of DNAJC6/AUXI, and PICALM, proteins that are risk factors for PD and/or AD respectively, were detected. We compared our study with two key prior O-GlcNAc proteome studies using mouse cerebral tissue and human AD brains. Among those identified to be increased by TG, 15 are also identified to be increased in human AD brains compared to control, including those involved in cytoskeleton, autophagy, chromatin organization and mitochondrial dysfunction. These studies provide insights regarding neurodegenerative diseases therapeutic targets.