Adiponectin is an adipokine with potent anti-inflammatory properties. Treatment of macrophages with adiponectin results in a suppression of lipopolysaccharide (LPS)-stimulated cytokine production. Here we investigated the transcriptional and post-transcriptional mechanisms by which adiponectin suppresses LPS-stimulated tumor necrosis factor (TNF)-alpha production. Treatment of RAW 264.7 macrophages with LPS increased TNF-alpha promoter-driven luciferase activity (TNF-alpha promoter/Luc activity) by 20-fold over basal. After culture with 1 mug/ml globular adiponectin (gAcrp) for 18 h, TNF-alpha promoter/Luc activity was increased even in the absence of LPS; further challenge with LPS only increased TNF-alpha promoter/Luc activity by 1.4-fold. Treatment with gAcrp decreased LPS-stimulated ERK1/2 phosphorylation and IkappaB degradation and suppressed the ability of LPS to increase the DNA binding activity of Egr-1 and p65. gAcrp also suppressed LPS-mediated stabilization of TNF-alpha mRNA. In controls cells, the half-life of TNF-alpha mRNA was increased from approximately 30 min at base line to approximately 80 min in response to LPS. After treatment with gAcrp for 18 h, LPS failed to increase TNF-alpha mRNA stability. This gAcrp-mediated loss of stimulus-induced stabilization of TNF-alpha mRNA required the presence of the TNF-alpha 3'-untranslated region and was associated with an increase in expression and RNA binding activity of tristetraprolin, an mRNA-binding protein that destabilizes TNF-alpha mRNA. In summary, these data characterize the complex transcriptional and post-transcriptional effects of gAcrp on LPS-stimulated TNF-alpha expression in macrophages. gAcrp treatment profoundly suppressed the ability of LPS to increase TNF-alpha transcription and reduced the stimulus-induced stabilization of TNF-alpha mRNA in response to LPS.