O-linked β-N-acetylglucosmaine (O-GlcNAc) is an essential, ubiquitous, dynamic modification of metazoan nucleocytoplasmic proteins. Unlike prototypical glycosylation, O-GlcNAc is not elongated into more complex structures and it is localized almost exclusively to nuclear and cytoplasmic proteins. O-GlcNAc modifies Ser / Thr residues in peptide motifs either identical or similar to those used by kinases. In some instances, O-GlcNAc and phosphorylation occur at the same site, suggesting that a complex interplay exists between these post-translational modifications. Deletion of the gene that adds O-GlcNAc to the protein backbone, the UDP-GlcNAc: polypeptide O-β-Nacetylglucosaminyltransferase, is lethal at the single cell level underlying the importance of O-GlcNAc. O-GlcNAc is rapidly emerging as a key nutrient sensor regulating signaling, transcription and cellular responses to stress.