O-linked -acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification found on hundreds of nucleocytoplasmic proteins in metazoa. Although a single enzyme， O-GlcNAc transferase (OGT)， generates the entire cytosolic O-GlcNAc proteome， it is not understood how it recognizes its protein substrates， targeting only a fraction of serines/threonines in the metazoan proteome for glycosylation. We describe a trapped complex of human OGT with the C-terminal domain of TAB1， a key innate immunity-signalling O-GlcNAc protein， revealing extensive interactions with the tetratricopeptide repeats of OGT. Confirmed by mutagenesis， this interaction suggests that glycosylation substrate specificity is achieved by recognition of a degenerate sequon in the active site combined with an extended conformation C-terminal of the O-GlcNAc target site.