O-GlcNAc transferase (OGT) is essential for viability of all mammalian cells but no one knows why. OGT has two catalytic activities that take place in the same active site. In one, OGT attaches N-acetyl glucosamine to serine and threonine side chains of at least a thousand nuclear and cytoplasmic proteins. In the other, OGT cleaves a transcriptional co-activator involved in cell-cycle regulation. Because OGT is found in several stable protein-complexes, it is also proposed to act as a scaffolding protein. How do you dissect the cellular functions of an essential protein that has multiple biochemical activities and a thousand different substrates? I will argue that you need to start by understanding the chemistry – mechanisms of catalysis and substrate recognition – so you can develop variants with some activities and not others. I will describe how we have done so and how we have used our knowledge to answer a fundamental question: What is the essential biochemical function of OGT for cell survival? I will also describe how we have developed and used a specific small molecule inhibitor of OGT to uncover an unexpected role in controlling RNA splicing.