O-Linked beta-N-acetylglucosaminylation (O-GlcNAcylation) of nucleocytoplasmic proteins is a ubiquitous post-translational modification in multicellular organisms studied so far. Since aberrant O-GlcNAcylation has a link with insulin resistance, iris important to establish the status of O-GIcNAcylation in differentiation of mesenchymal cells such as preadipocytes. In this study, we found a differentiation-dependent drastic increase in the level of O-GIcNAcylation in mouse 3T3-L1 preadipocytes. The occurrence of the increase in O-GIcNAcylation, which correlated with the expression of C/EBP alpha, was in part due to increased expression of O-GlcNAc transferase. In addition to the well-known O-GlcNAcylated proteins such as nucleoporins and vimentin, pyruvate carboxylase, long chain fatty acid-CoA ligase 1, and Ewing sarcoma protein were identified as the proteins which are heavily O-GlcNAcylated with the adipocyte differentiation. Both adipocyte differentiation and the differentiation-dependent increase in O-GIcNAcylation were blocked by 6-diazo-5-oxo-norleucine. These results suggest that O-GIcNAcylation particilates, at least in part, in adipogenesis. (C) 2010 Elsevier Inc. All rights reserved.