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Despite the known influence of DNA methylation from lipopolysaccharide (LPS) activation, data on the O6-methylguanine-DNA methyltransferase (MGMT, a DNA suicide repair enzyme) in macrophages is still lacking. The transcriptomic profiling of epigenetic enzymes from wild-type macrophages after single and double LPS stimulation, representing acute inflammation and LPS tolerance, respectively, was performed. Small interfering RNA (siRNA) silencing of <i>mgmt</i> in the macrophage cell line (RAW264.7) and <i>mgmt</i> null (<i>mgmt</i>^flox/flox; LysM-Cre^cre/−) macrophages demonstrated lower secretion of TNF-α and IL-6 and lower expression of pro-inflammatory genes (<i>iNOS</i> and <i>IL-1β</i>) compared with the control. Macrophage injury after a single LPS dose and LPS tolerance was demonstrated by reduced cell viability and increased oxidative stress (dihydroethidium) compared with the activated macrophages from littermate control mice (<i>mgmt</i>^flox/flox; LysM-Cre^−/−). Additionally, a single LPS dose and LPS tolerance also caused mitochondrial toxicity, as indicated by reduced maximal respiratory capacity (extracellular flux analysis) in the macrophages of both <i>mgmt</i> null and control mice. However, LPS upregulated <i>mgmt</i> only in LPS-tolerant macrophages but not after the single LPS stimulation. In mice, the <i>mgmt</i> null group demonstrated lower serum TNF-α, IL-6, and IL-10 than control mice after either single or double LPS stimulation. Suppressed cytokine production resulting from an absence of <i>mgmt</i> in macrophages caused less severe LPS-induced inflammation but might worsen LPS tolerance.