Dynamic modification of proteins with O-GlcNAc modification at serine and threonine residues of proteins is carried out by OGT and removal of O-GlcNAc is carried out by OGA. O-GlcNAc levels respond to nutrient availability including glucose, which is assimilated via the hexosamine biosynthetic pathway (HBSP) to form uridine diphospho-N-acetylglucosamine (UDP-O-GlcNAc). The mechanisms and structures of these enzymes are being uncovered as is the basis for the substrate specificity of these enzymes. Display omitted ► O-GlcNAc levels in cells respond to availability of nutrients including glucose. ► OGT and OGA are the two enzymes processing hundreds of O-GlcNAc modified proteins. ► Catalysis by OGA and OGT is being studied using kinetic and structural approaches. ► OGA processes metabolic derivatives of O-GlcNAc from proteins. ► The substrate specificity and regulation of OGA and OGT merit further attention. The addition of N-acetylglucosamine (GlcNAc) O-linked to serine and threonine residues of proteins is known as O-GlcNAc. This post-translational modification is found within multicellular eukaryotes on hundreds of nuclear and cytoplasmic proteins. O-GlcNAc transferase (OGT) installs O-GlcNAc onto target proteins and O-GlcNAcase (OGA) removes O-GlcNAc. Their combined action makes O-GlcNAc reversible and serves to regulate cellular O-GlcNAc levels. Here I review select recent literature on the catalytic mechanism of these enzymes and studies on the molecular basis by which these enzymes identify and process their substrates. Molecular level understanding of how these enzymes work, and the basis for their specificity, should aid understanding how O-GlcNAc contributes to diverse cellular processes ranging from cellular signaling through to transcriptional regulation.