Abstract Human O-GlcNAcase (OGA) regulates a wide variety of essential biological processes by hydrolyzing O- GlcNAcylation from numerous proteins in response to nutrient and stress. Even though dysregulated OGA has been indicated in many diseases, little is known about how OGA's function is regulated. Dynamic function of OGA is mainly reflected by the changes of its substrate specificity, which is challenging to be characterized. One part of the challenge is the lack of information on OGA substrate recognition, as no apparent sequence motif can be defined in its substrate proteins. The other part of the challenge is the paucity of strategies to investigate OGA's substrate targeting and regulatory mechanism in cells. As a breakthrough in the field, we recently solved the first crystal structures of human OGA, revealing a unique structural feature that could contribute to substrate recognition. We propose to further characterize OGA's substrate specificity by solving new structures of OGA in complex with various substrates. We also propose to develop an innovative strategy to profile OGA's physiological substrates and binding partners in an effort to uncover the molecular signatures underlying the functional dysregulation of OGA in cancer development and programmed cell death. This study will provide unprecedented insights into the critical role of OGA in coupling O-GlcNAc biology to modulation of cell survival and death.