O-GlcNAcylation, a post-translational modification consisting of O-linked N-acetylglucosamine attached to serine and threonine residues, occurs in thousands of cytoplasmic, nuclear, and mitochondrial proteins but has been reported for relatively few viral proteins. We used click chemistry, specific antibodies, and mass spectrometry to investigate the O-GlcNAcylation of vaccinia virus (VACV) proteins. A virion protein of ~40 kDa was identified by SDS-polyacrylamide gel electrophoresis following azide-alkyne cycloaddition of biotin or an infrared dye to O-GlcNAc residues. Candidate O-GlcNAc virion proteins were detected by mass spectrometry, and A4, a highly conserved core component required for virion assembly, was identified by decreased electrophoretic mobility resulting from the specific attachment of multiple 10 kDa polyethylene glycol residues to O-GlcNAc sites. O-GlcNAc was not detected in virions of an A4 deletion mutant, suggesting A4 is the only or major constituent with this modification. Multiple O-GlcNAc modified amino acids in intrinsically disordered regions of A4 were identified by electron transfer dissociation mass spectrometry. Recombinant A4 was O-GlcNAcylated following stable and transient transfection of uninfected cell lines, suggesting a role for a cellular enzyme, which was confirmed by reduction of the modification by specific inhibitors of O-GlcNAc transferase during virus infection. Moreover, induced degradation of O-GlcNAc transferase prior to VACV infection decreased O-GlcNAcylation of A4 to undetectable levels without diminishing the A4 abundance. Nevertheless, the specific infectivity of O-GlcNAc-deficient virus particles was unimpaired. O-GlcNAcylation either has a subtle role in the VACV life cycle, or A4 is an inadvertent substrate of the promiscuous O-GlcNAc transferase.IMPORTANCEO-GlcNAc is a reversible enzymatic post-translational modification of serine and threonine residues found on thousands of cellular proteins with roles in regulating numerous functions including signal transduction, transcription, and stress response. However, little is known about O-GlcNAc modifications of viral proteins. Here, we report that the vaccinia virus A4 core protein has multiple O-GlcNAc modifications. The cellular O-GlcNAc transferase was shown to be required for modifying the vaccinia virus protein, which is synthesized and assembled into virus particles within cytoplasmic virus factories. Moreover, inhibition and degradation of the transferase prevented O-GlcNAcylation of A4. Nevertheless, virus assembly and replication in vitro were unaffected by the absence of the modification, suggesting that the addition of O-GlcNAc to A4 has a subtle role or that the modification is a byproduct of a promiscuous O-GlcNAc transferase that preferentially modifies intrinsically disordered regions of proteins.