The pancreatic/duodenal homeobox-1 protein (PDX-1, also called STF-1, IPF-1) is a transcription factor that plays an important role in pancreatic function and development. Here, we have overexpressed and purified PDX-1 from baculovirus/sf-9 cells, transiently transfected Cos-7 cells and native Min6 cells and demonstrated that the protein is posttranslationally modified by O-linked N-acetylglucosamine (O-GlcNAc). The approaches we used include binding of the protein to the lectin WGA, labeling with galactosyltransferase and UDP-[(3)H]gal and probing with the O-GlcNAc-specific antibody, RL-2. PNGase F treatment and structural analysis indicate that the carbohydrate is beta-linked O-GlcNAc. Mapping of [(3)H]gal-labeled tryptic peptides indicates that PDX-1 has two major sites for O-GlcNAcylation. In Min6 cells, elevated glucose concentration leads to an increase in protein O-GlcNAcylation and this hyperglycosylation correlates with an increase in DNA binding activity of PDX-1 and insulin secretion. On the other hand, the GFAT inhibitor azaserine reduces intracellular O-GlcNAc levels and profoundly attenuates glucose-stimulated insulin secretion. These data suggest that O-GlcNAcylation may be involved in the regulation of PDX-1 DNA binding activity and in glucose-stimulated insulin secretion in beta-cells.