The intracellular modification of proteins by the addition of a single O-linked N-acetylglucosamine (O-GlcNAc) molecule is a ubiquitous post-translational modification in eukaryotic cells. It is catalysed by O-linked N-acetylglucosaminyltransferase, which attaches O-GlcNAc to serine/threonine residues, and it is counter-regulated by beta-N-acetylglucosaminidase, which is the antagonistic glycosidase that removes the O-GlcNAc group. O-GlcNAc modification competes with phosphorylation by protein kinases at similar sites, thereby affecting important signalling nodes. Accumulating evidence supports a central role for O-GlcNAc modifications and the corresponding enzymes in the regulation of immune cells, particularly in the activation processes of T and B lymphocytes. Here, we discuss recent advances in the field of O-GlcNAc modifications, focusing on the cells of the immune system.