The peroxisome proliferator-activated receptor gamma (PPAR gamma), a member of the nuclear receptor superfamily, is a key regulator of adipogenesis and is important for the homeostasis of the adipose tissue. The beta-O-linked N-acetylglucosamine (O-GlcNAc) modification, a posttranslational modification on various nuclear and cytoplasmic proteins, is involved in the regulation of protein function. Here, we report that PPAR gamma is modified by O-GIcNAc in 3T3-L1 adipocytes. Mass spectrometric analysis and mutant studies revealed that the threonine 54 of the N-terminal AF-1 domain of PPAR gamma is the major O-GlcNAc site. Transcriptional activity of wild type PPAR gamma was decreased 30% by treatment with the specific O-GlcNAcase (OGA) inhibitor, but the T54A mutant of PPAR gamma did not respond to inhibitor treatment. In 3T3-L1 cells, an increase in O-GlcNAc modification by OGA inhibitor reduced PPAR gamma transcriptional activity and terminal adipocyte differentiation. Our results suggest that the O-GlcNAc state of PPAR gamma influences its transcriptional activity and is involved in adipocyte differentiation. (C) 2012 Elsevier Inc. All rights reserved.