O-linked-N-acetylglucosaminylation (O-GlcNAcylation) is a type of glycosylation that occurs when a monosaccharide, O-GlcNAc, is added onto serine or threonine residues of nuclear or cytoplasmic proteins by O-GlcNAc transferase (OGT) and which can be reversibly removed by O-GlcNAcase (OGA). O-GlcNAcylation couples the processes of nutrient sensing, metabolism, signal transduction and transcription, and plays important roles in development, normal physiology and physiopathology. Cumulative studies have indicated that O-GlcNAcylation affects the functions of protein substrates in a number of ways, including protein cellular localization, protein stability and protein/protein interaction. Particularly, O-GlcNAcylation has been shown to have intricate crosstalk with phosphorylation as they both modify serine or threonine residues. Aberrant O-GlcNAcylation on various protein substrates has been implicated in many diseases, including neurodegenerative diseases, diabetes and cancers. However, the role of protein O-GlcNAcylation in immune cell lineages has been less explored. This review summarizes the current understanding of the fundamental biochemistry of O-GlcNAcylation, and discusses the molecular mechanisms by which O-GlcNAcylation regulates the development, maturation and functions of immune cells. In brief, O-GlcNAcylation promotes the development, proliferation, and activation of T and B cells. O-GlcNAcylation regulates inflammatory and antiviral responses of macrophages. O-GlcNAcylation promotes the function of activated neutrophils, but inhibits the activity of nature killer cells.