Abstract The essential human enzyme O-GlcNAc transferase (OGT) catalyzes a unique type of intracellular protein glycosylation called O-GlcNAcylation. In response to nutrient levels and stress, OGT dynamically regulates a variety of physiological and pathological processes including the “Warburg effect” in cancer cells and insulin resistance in diabetes. Previous studies on the OGT active site have made fundamental discoveries on its catalytic mechanism and substrate interactions. However, how OGT regulates protein- and site-specific O- GlcNAcylation remains unclear. This is due to a number of challenges including: 1) OGT glycosylates thousands of proteins without a conserved sequence motif near the O-GlcNAc modification site, 2) a majority of O- GlcNAcylation sites are found on intrinsically disordered regions (IDRs), 3) OGT typically binds proteins with low/moderate affinity, and 4) a lack of OGT-protein complex structures. In our last funding period, we have made strides in these areas through development of a suite of novel chemical probes that allow us to interrogate OGT specific interactions with low/moderate affinity for structural, proteomic, and biochemical characterizations. This proposal aims to make further conceptual and technical breakthroughs toward addressing these longstanding challenges. It is expected that a better understanding of how OGT interacts with other proteins, particularly through the regions beyond the OGT catalytic site, will be essential for understanding OGT’s functional regulation at protein- and site-specific levels, filling major knowledge gaps between decades of biological observations of OGT’s nutrient sensing and other regulatory roles, and will support the need to specifically modulate OGT functions for biomedical applications.