Treatment of macrophages with lipopolysaccharide (LPS), a major component of the Gram-negative bacteria outer membrane, results in elevated O-GlcNAc in cellular and mouse models. During infection, cytosolic OGT was de-S-nitrosylated and activated. Suppressing O-GlcNAc with DON inhibited the O-GlcNAcylation and nuclear translocation of NF-κB, as well as downstream induction of inducible nitric oxide synthase and IL-1β production. DON treatment also improved cell survival during LPS treatment.