Many eukaryotic proteins contain O-linked N-acetylglucosamine (O-GlcNAc) on their serine and threonine side chain hydroxyls. In contrast to classical cell surface glycosylation, O-GlcNAc occurs on resident nuclear and cytoplasmic proteins. O-GlcNAc exists as a single monosaccharide residue, showing no evidence of further elongation. Like phosphorylation, O-GlcNAc is highly dynamic, transiently modifying proteins. These post-translational modifications give rise to functionally distinct subsets of a given protein. Furthermore, all known O-GlcNAc proteins are also phosphoproteins that reversibly form multimeric complexes that are sensitive to the state of phosphorylation. This observation implies that O-GlcNAc may work in concert with phosphorylation to mediate regulated protein interactions. The proteins that bear the O-GlcNAc modification are very diverse, including RNA polymerase II and many of its transcription factors, numerous chromatin-associated proteins, nuclear pore proteins, proto-oncogenes, tumor suppressors and proteins involved in translation. Here, we discuss the functional implications of O-GlcNAc-modifications of proteins involved in various aspects of gene expression, beginning with proteins involved in transcription and ending with proteins involved in regulating protein translation.