O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT), which catalyzes the addition of a single β-N-GlcNAc unit to target proteins, has been shown to act as a transcriptional regulator. In the current study, we discovered that OGT exerted inhibitory effects on the LPS-driven activation of NF-B and iNOS. In response to LPS, OGT exhibited an increased interaction with the transcriptional corepressor, mSin3A. Furthermore, mSin3A, HDAC1, and HDAC2 displayed increased binding to the iNOS promoter in response to LPS. Treatment with GlcN, in contrast, inhibits LPS-induced inflammation and decreased LPS-mediated recruitment of OGT, mSin3A and HDACs. LPS treatment also resulted in the hypo-O-GlcNAcylation of mSin3A, which was reversed by GlcN. When the effect of the HDAC inhibitor, trichostatin A (TSA), on LPS- and/or GlcN-mediated iNOS protein/mRNA induction was investigated, the results revealed that TSA dose-dependently enhanced iNOS expression in response to LPS and/or GlcN. In addition, histone acetyltransferases (HATs), p300 and CBP enhanced LPS- and/or GlcN-induced iNOS protein expression. These results collectively suggest that OGT inhibits LPS-driven NF-B activation and subsequent iNOS transcription by modulating histone acetylation either directly or indirectly.