Nicotine inhibits the release of TNF-α from macrophage through activation of STAT3. Tristetraprolin (TTP) is known to destabilize pro-inflammatory transcripts containing AU-rich elements (ARE) in 3′-untranslated region (3′-UTR). Here we show that in LPS-stimulated human macrophages the anti-inflammatory action of nicotine is mediated by TTP. Nicotine induced activation of STAT3 enhanced STAT3 binding to the TTP promoter, increased TTP promoter activity, and increased TTP expression resulting in the suppression of LPS-stimulated TNF-α production. Overexpression of a dominant negative mutant of STAT3 (R382W) or down-regulation of STAT3 by siRNA abolished nicotine-induced TTP expression and suppression of LPS-stimulated TNF-α production. Nicotine enhanced the decay of TNF-α mRNA and decreased luciferase expression of a TNF-α 3′-UTR reporter plasmid in U937 cells. However, siRNA to TTP abrogated these effects of nicotine. In this experiment, we are reporting for the first time the involvement of TTP in the cholinergic anti-inflammatory cascade consisting of nicotine-STAT3-TTP- dampening inflammation.