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 ofmgmtin the macrophage cell line (RAW264.7) andmgmtnull (mgmtflox/flox; LysM-Crecre/−) macrophages demonstrated lower secretion of TNF-α and IL-6 and lower expression of pro-inflammatory genes (iNOSandIL-1β) 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 (mgmtflox/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 bothmgmtnull and control mice. However, LPS upregulatedmgmtonly in LPS-tolerant macrophages but not after the single LPS stimulation. In mice, themgmtnull 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 ofmgmtin macrophages caused less severe LPS-induced inflammation but might worsen LPS tolerance.