O-linked N-acetylglucosamine (O-GlcNAc) was first discovered 30 years ago and is now recognized as an abundant form of protein O-glycosylation found in the nucleus and cytoplasm of multicellular eukaryotes. This modification is found on hundreds of proteins yet is regulated by only two enzymes; O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAc is in some cases reciprocal to serine/threonine phosphorylation and its levels fluctuate in response to nutrient availability and cellular stress. Consistent with the metabolic responsiveness of O-GlcNAc, this modification has been implicated in various essential biological processes ranging from regulation of gene expression through to circadian rhythm. Accordingly, inappropriate regulation of O-GlcNAc has been proposed to play roles in diverse diseases including various cancers and Alzheimer disease. Here I will discuss research from our laboratory focused on understanding the substrate specificities and catalytic mechanisms of OGT and OGA, as well as the exploitation of these insights to design chemical probes of these enzymes that are active in cells and tissues. I will also describe how we have recently used these tools to define the functional roles of O-GlcNAc in various cellular processes including proteostasis as well as in animal models of diseases including Alzheimer disease.