Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a typical glycolytic enzyme comprised of four identical 37 kDa subunits. In addition to its glycolytic function, GAPDH has a number of biological functions which are related to its subcellular localization. Generally, protein O-linked N-acetylglucosamine modification (O-GlcNAcylation) is considered, among other effects, to mediate the nuclear transportation of cytosolic proteins. To elucidate the effect of O-GlcNAcylation on GAPDH, we determined the location of the O-GlcNAcylation site by tandem mass spectrometry, and subsequently examined the biological significance of this derivatization. The site involved was identified to be Thr227 by beta-elimination and Michael addition. Transient transfection assays demonstrated that the T227A mutation induced the cytoplasmic accumulation of GAPDH, whereas the wild type was present in the cytoplasm and nuclei. Structural modeling, mutagenesis of Thr227 to Lys and Arg, and gel filtration chromatography of mutated and wild type GAPDH, together suggested that O-GlcNAcylation at Thr227 interrupts the hydrophobic interfaces formed between GAPDH monomers in its tetrameric state. The present study identified Thr227 as the major GAPDH O-GlcNAcylation site, which suggests that this modification mediates the nuclear translocation of GAPDH, presumably by disrupting the conformation of tetrameric GAPDH.