Harmine is a beta-carboline alkaloid present in various plants, including in the seeds of Peganum harmala L. This study aimed to investigate the anti-inflammatory activity and mechanism of harmine using macrophages stimulated with various toll-like receptor (TLR) agonists and a model of endotoxemia. The expression of inflammatory mediators induced by ligands of TLRs 2, 3, 4, and 9 were examined in thioglycollate-elicited peritoneal macrophages isolated from BALB/c and C57BL/6 mouse strains. Further, the activation of NF-kappaB, MAPK, AP-1, and STAT1 was explored using lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly(I:C)). Finally, the liver inflammatory response during endotoxemia was examined. Harmine inhibited inducible nitric oxide synthase, cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), IL-12, and other markers induced by various TLR agonists. The inhibition of NF-kappaB activity by harmine occurred via the modulation of p65 phosphorylation, independent of IkappaBalpha degradation. The inhibition of AP-1 activity by harmine was associated with the modulation of JNK. Harmine inhibited the LPS-induced serine and tyrosine phosphorylation of STAT1, but only affected serine phosphorylation by poly(I:C) treatment. In vivo, harmine inhibited iNOS and COX-2 expression during endotoxemia. Collectively, the results show that harmine can be effective against infectious inflammation through modulation of NF-kappaB, JNK, and STAT1.