ABSTRACTO-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential enzyme that modifies proteins withO-GlcNAc. InbornOGTgenetic variants were recently shown to mediate a novel type of congenital disorder of glycosylation (OGT-CDG), which is characterised by X-linked intellectual disability (XLID) and developmental delay. Here, we report an OGTC921Yvariant that co-segregates with XLID and epileptic seizures, and results in loss of catalytic activity. Colonies formed by mouse embryonic stem cells carrying OGTC921Yshowed decreased levels of proteinO-GlcNAcylation accompanied by decreased levels of Oct4 (encoded byPou5f1), Sox2 and extracellular alkaline phosphatase (ALP), implying reduced self-renewal capacity. These data establish a link between OGT-CDG and embryonic stem cell self-renewal, providing a foundation for examining the developmental aetiology of this syndrome.