We have previously demonstrated that in a rat model of trauma-hemorrhage (T-H), glucosamine administration during resuscitation improved cardiac function, reduced circulating levels of inflammatory cytokines, and increased tissue levels of O-linked W-acetylglucosamine (O-GlcNAc) on proteins. The mech- anism(s) by which glucosamine mediated its protective effect were not determined; therefore, the goal of this study was to test the hypothesis that glucosamine treatment attenuated the activation of the nuclear factor-κB (NF-κB) signaling pathway in the heart via an increase in protein O-GlcNAc levels. Fasted male rats were subjected to T-H by bleeding to a mean arterial blood pressure of 40 mmHg for 90 min followed by resuscitation. Glucosamine treatment during resuscitation significantly attenuated the T-H-induced increase in cardiac levels of TNF-α and IL-6 mRNA, IκB-α phosphorylation, NF-κB, NF-κB DNA binding activity, ICAM-1, and MPO activity. LPS (2 μg/ml) increased the levels of IκB-α phosphorylation, TNF-α, ICAM-1, and NF-κB in primary cultured cardiomyocytes, which was significantly attenuated by glucosamine treatment and overexpression of O-GlcNAc transferase; both interventions also significantly increased O-GlcNAc levels. In contrast, the transfection of neonatal rat ventricular myocytes with OGT small-interfering RNA decreased O-GlcNAc transferase and O-GlcNAc levels and enhanced the LPS- induced increase in IκB-α phosphorylation. Glucosamine treatment of macrophage cell line RAW 264.7 also increased O-GlcNAc levels and attenuated the LPS-induced activation of NF-κB. These results demonstrate that the modulation of O-GlcNAc levels alters the response of cardiomyocytes to the activation of the NF-κB pathway, which may contribute to the glucosamine-mediated improvement in cardiac function following hemorrhagic shock. Copyright © 2009 the American Physiological Society.