O-linked N-acetylglucosamine (O-GlcNAc), a monosaccharide N-acetylglucosamine addition on nucleocytoplasmic proteins, is abundant in transcription regulators and has been implicated in gene regulation. Sp1 transcription factor is multiply modified by O-GlcNAc within its serine/threonine-rich region and glutamine-rich transactivation domain. In the present study, we show that O-GlcNAc of Sp1 serine/threonine-rich region interrupts a physical interaction between Sp1 and NF-YA, thus inhibiting Sp1-NF-Y cooperative activation of gene transcription. Our results strengthen the notion that O-GlcNAc regulates gene transcription by modulating the protein-protein interaction network among transcription regulatory proteins.