Polycomb group proteins are conserved transcriptional repressors that control animal and plant development. Here, we found that the Drosophila Polycomb group gene super sex combs (sxc) encodes Ogt, the highly conserved glycosyltransferase that catalyzes the addition of N-acetylglucosamine (GlcNAc) to proteins in animals and plants. Genome-wide profiling in Drosophila revealed that GlcNAc-modified proteins are highly enriched at Polycomb response elements. Among different Polycomb group proteins, Polyhomeotic is glycosylated by Sxc/Ogt in vivo. sxc/Ogt-null mutants lacked O-linked GlcNAcylation and failed to maintain Polycomb transcriptional repression even though Polycomb group protein complexes were bound at their target sites. Polycomb repression appears to be a critical function of Sxc/Ogt in Drosophila and may be mediated by the glycosylation of Polyhomeotic.