Human O-GlcNAcase (OGA) is the sole enzyme that hydrolyzes O-GlcNAcylation, an essential intracellular protein glycosylation that functionally regulates thousands of proteins in response to nutrients and stress. Aberrant functions of OGA have been detected in numerous diseases including cancer and neurodegeneration, fostering intense interest in OGA as a therapeutic target. However, due to the lack of complete OGA structure and the paucity of information on its substrate recognition, little is known about how OGA can accommodate such a broad range of substrates without a consensus sequence motif, while still maintaining certain level of specificity. Our preliminary studies provide strong evidence suggesting that OGA’s non-catalytic regions play critical roles in substrate binding and new functions that we are just beginning to reveal. We propose to advance structural and functional investigation of OGA. The new findings from this study will help decode OGA’s complex cellular regulation and will serve as a critical foundation for manipulating the precise role of OGA, paving the way for development of more effective and safer therapies for challenging diseases.