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Protein O-GlcNAcylation is an abundant, dynamic and reversible type of protein post-translational modification in animals that has been implicated in signalling processes linked to innate immunity, stress response, growth factor response, transcription, translation and proteosomal degradation. Only two enzymes, O-GlcNAc (O-linked N-acetylglucosamine) transferase and O-GlcNAcase, catalyse the reversible addition of the O-GlcNAc residue to over 1000 target proteins in the human cell. Recent advances in our understanding of the structures and mechanisms of these enzymes have resulted in the development of potent and selective inhibitors. The present review gives an overview of these inhibitors and how they have been used on cell lines, primary cells and animals to modulate O-GlcNAc levels and study the effects on signal transduction.