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ETHNOPHARMACOLOGICAL RELEVANCE Dianthi Herba is a traditional herbal medicine used to treat inflammatory-related diseases including acute pyelonephritis, cystitis, laryngopharyngitis, and urethritis. AIM OF THE STUDY We investigated the effects of Dianthi Herba ethanolic extract (DH) on lipopolysaccharide (LPS)-mediated inflammatory responses in murine macrophages including RAW 264.7 cell line and mouse peritoneal macrophages as well as nociceptive and edema mouse models. MATERIALS AND METHODS The biological effects of DH on inflammatory cytokine, mediator, and related protein production were assessed using enzyme-linked immunosorbent assay (ELISA), Western blotting, and real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). Additionally, Western blotting was performed to investigate intracellular signaling pathways, and the anti-nociceptive activity of three doses of DH (100, 200, and 300mg/kg) against acetic acid-induced writhing responses and its inhibitory effects on xylene-induced ear edema were researched in mice through oral administration. RESULTS DH treatment significantly inhibited nitric oxide (NO) secretion and inflammatory cytokine production in RAW 264.7 cells and mouse peritoneal macrophages and induced heme oxygenase (HO)-1 expression. DH strongly inhibited the transcriptional activity of nuclear factor (NF)-κB and phosphorylation of mitogen-activated protein kinases (MAPK) in LPS-stimulated macrophages. Meanwhile, DH exerted anti-nociceptive effects on writhing responses and anti-edema effects in mice. CONCLUSION We confirmed the anti-inflammatory activities and inhibitory mechanism of DH in macrophages and clarified its inhibitory effects in vivo. These findings illustrate the therapeutic potential of DH as a natural anti-inflammatory agent.