O‐Linked N‐acetylglucosamine (O‐GlcNAc) modification of proteins plays an important role in transcription, translation, nuclear transport and cytoskeletal assembly. We have shown that glucosamine (GlcN) increased O‐GlcNAc levels and attenuated activation of NF‐κB signaling following trauma hemorrhage and resuscitation in vivo and in response to lipopolysaccharide (LPS) treatment of in cardiomyocytes in vitro. Therefore the goal of this study was to determine whether the effect of GlcN on NF‐κB activation was mediated via O‐GlcNAc transferase (OGT), which catalyzes O‐GlcNAc synthesis. Neonatal rat ventricular myocytes(NRVMs) were untreated, treated with 5mM GlcN, transfected with OGT adenovirus or OGT siRNA and then stimulated with LPS (2μg/ml) for 6h. In untreated cells LPS significantly increased IκB‐α phosphorylation, TNF‐α and ICAM1 expression and increased nuclear NF‐κB levels. Both GlcN and OGT overexpression significantly increased O‐GlcNAc levels, attenuated LPS‐induced TNF‐α and ICAM1 expression and decreased IκB‐α phosphorylation and nuclear NF‐κB levels. In contrast, OGT siRNA treatment decreased OGT and O‐GlcNAc levels and enhanced LPS‐induced increase in IκB‐α phosphorylation. These results demonstrate that modulation of cellular O‐GlcNAc levels alters the response to activation of NF‐κB signaling pathway, and may contribute the protective effect of GlcN in vivo.