The O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) mediates intracellular O-GlcNAcylation modification, whose function and substrates have entranced biologists and chemists alike. O-GlcNAcylation occurs on Ser/Thr residues and takes part in a vast array of physiological processes. OGT is essential for dividing mammalian cells, and it underscores many human diseases. Yet many of its fundamental substrates in the cell division process remains to be unveiled. Here we focus on its effect on Polo-like kinase 1 (PLK1), a mitotic master kinase that governs DNA replication, mitotic entry, chromosome segregation and mitotic exit. We found that PLK1 interacts with OGT and is O-GlcNAcylated.