Updated project metadata. O-GlcNAcylation is a dynamic and reversible post-translational modification involving the attachment of a single β-N-acetylglucosamine (GlcNAc) moiety to the hydroxyl groups of serine or threonine residues. This modification occurs in thousands of cytoplasmic, nuclear, and mitochondrial proteins in mammalian cells and plays critical roles in regulating protein function, localization, and interactions. However, despite its widespread occurrence in host proteins, relatively few examples of O-GlcNAcylation have been reported in viral proteins. In this study, we sought to investigate the presence and functional relevance of O-GlcNAcylation in the vaccinia virus (VACV), a model poxvirus with a well-characterized replication cycle and virion architecture.