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<i>Glossogyne tenuifolia</i> Cassini (<i>Hsiang-Ju</i> in Chinese) is a perennial herb native to Taiwan. It was used in traditional Chinese medicine (TCM) as an antipyretic, anti-inflammatory, and hepatoprotective agent. Recent studies have shown that extracts of <i>G. tenuifolia</i> possess various bioactivities, including anti-oxidant, anti-inflammatory, immunomodulation, and anti-cancer properties. However, the pharmacological activities of <i>G. tenuifolia</i> essential oils have not been studied. In this study, we extracted essential oil from air-dried <i>G. tenuifolia</i> plants, then investigated the anti-inflammatory potential of <i>G. tenuifolia</i> essential oil (GTEO) on lipopolysaccharide (LPS)-induced inflammation in murine macrophage cells (RAW 264.7) in vitro. Treatment with GTEO (25, 50, and 100 μg/mL) significantly as well as dose-dependently inhibited LPS-induced pro-inflammatory molecules, such as nitric oxide (NO) and prostaglandin E₂ (PGE₂) production, without causing cytotoxicity. Q-PCR and immunoblotting analysis revealed that the inhibition of NO and PGE₂ was caused by downregulation of their corresponding mediator genes, inducible nitric oxide synthase (<i>iNOS</i>), and cyclooxygenase-2 (<i>COX-2</i>), respectively. Immunofluorescence and luciferase reporter assays revealed that the inhibition of <i>iNOS</i> and <i>COX-2</i> genes by GTEO was associated with the suppression of nuclear export and transcriptional activation of the redox-sensitive transcription factor, nuclear factor -κB (NF-κB). In addition, GTEO treatment significantly inhibited phosphorylation and proteosomal degradation of the inhibitor of NF-κB (I-κBα), an endogenous repressor of NF-κB. Moreover, treatment with GTEO significantly blocked the LPS-mediated activation of inhibitory κB kinase α (IKKα), an upstream kinase of the I-κBα. Furthermore, <i>p</i>-cymene, β-myrcene, β-cedrene, <i>cis</i>-β-ocimene, α-pinene, and _D-limonene were represented as major components of GTEO. We found that treatment with <i>p</i>-cymene, α-pinene, and _D-limonene were significantly inhibiting LPS-induced NO production in RAW 264.7 cells. Taken together, these results strongly suggest that GTEO inhibits inflammation through the downregulation of NF-κB-mediated inflammatory genes and pro-inflammatory molecules in macrophage cells.