O-GlcNAcylation is a ubiquitous and dynamic post-translational modification on serine/threonine residues of nucleocytoplasmic proteins in metazoa, which plays a critical role in numerous physiological and pathological processes. But the O-GlcNAcylation on most proteins is often substoichiometric, which hinders the functional study of the O-GlcNAcylation. This study aimed to improve the production of highly O-GlcNAcylated recombinant proteins in Escherichia coli (E. coli). To achieve this goal, we constructed a bacterial artificial chromosome-based chloramphenicol-resistant expression vector co-expressing O-GlcNAc transferase (OGT) and key enzymes (phosphoglucose mutase, GlmM and N-acetylglucosamine-1-phosphate uridyltransferase, GlmU) of the uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis pathway in E. coli, which can effectively increase the O-GlcNAcylation of the OGT target protein expressed by another vector. The results revealed that the expression of GlmM and GlmU increases the cellular concentration of UDP-GlcNAc in E. coli, which markedly enhanced the activity of the co-expressed OGT to its target proteins, such as H2B, p53 and TAB1. Altogether, we established a widely compatible E. coli expression system for producing highly O-GlcNAcylated protein, which could be used for modifying OGT target proteins expressed by almost any commercial expression vectors in E. coli. This new expression system provides possibility for investigating the roles of O-GlcNAcylation in the enzymatic activity, protein-protein interaction and structure of OGT target proteins.