We previously showed that S‐adenosylmethionine (SAMe) and its metabolite methylthioadenosine (MTA) blocked lipopolysaccharide (LPS)‐induced tumor necrosis factor α (TNFα) expression in RAW (murine macrophage cell line) and Kupffer cells at the transcriptional level without affecting nuclear factor κ B nuclear binding. However, the exact molecular mechanism or mechanisms of the inhibitory effect were unclear. While SAMe is a methyl donor, MTA is an inhibitor of methylation. SAMe can convert to MTA spontaneously, so the effect of exogenous SAMe may be mediated by MTA. The aim of our current work is to examine whether the mechanism of SAMe and MTA's inhibitory effect on proinflammatory mediators might involve modulation of histone methylation. In RAW cells, we found that LPS induced TNFα expression by both transcriptional and posttranscriptional mechanisms. SAMe and MTA treatment inhibited the LPS‐induced increase in gene transcription. Using the chromatin immunoprecipitation assay, we found that LPS increased the binding of trimethylated histone 3 lysine 4 (H3K4) to the TNFα promoter, and this was completely blocked by either SAMe or MTA pretreatment. Similar effects were observed with LPS‐mediated induction of inducible nitric oxide synthase (iNOS). LPS increased the binding of histone methyltransferases Set1 and myeloid/lymphoid leukemia to these promoters, which was unaffected by SAMe or MTA. The effects of MTA in RAW cells were confirmed in vivo in LPS‐treated mice. Exogenous SAMe is unstable and converts spontaneously to MTA, which is stable and cell‐permeant. Treatment with SAMe doubled intracellular MTA and S‐adenosylhomocysteine (SAH) levels. SAH also inhibited H3K4 binding to TNFα and iNOS promoters. Conclusion: The mechanism of SAMe's pharmacologic inhibitory effect on proinflammatory mediators is mainly mediated by MTA and SAH at the level of histone methylation. (HEPATOLOGY 2008.)