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In order to establish an efficient method for separation of polymethoxyflavones (PMFs) and explore the anti-inflammatory mechanism of PMF monomers, a citrus variety rich in PMFs, Ougan (<i>Citrus reticulata</i> cv. <i>Suavissima</i>), was selected, and three monomers, including nobiletin, tangeretin, and 5-demethylnobiletin, were purified by ultrasonic-assisted extraction, solid phase extraction, and high-speed countercurrent chromatography separation. UPLC-MS was used to identify the three monomers. UPLC determined purities of 99.87% to nobiletin, 99.76% to tangeretin, and 98.75% to 5-demethylnobiletin with the standard curve method. A lipopolysaccharide (LPS)-induced NO releasing model was performed in the mouse microglia BV-2 cell line. Results illustrated that PMF monomers inhibited the NO release and the inflammation-related cytokines, including IL-1&#946;, IL-6, and TNF&#945; elevation. QRT-PCR revealed that PMFs alleviated LPS-induced upregulation of <i>iNOS</i>, <i>IL-6</i>, <i>JAK2</i>, <i>TNF&#945;</i>, <i>IL-1&#946;</i>, and <i>NF-&#954;B</i> and LPS-induced downregulation of <i>I&#954;B&#945;</i>, while they did not affect <i>TLR1</i>, <i>TLR2</i>, <i>TLR4</i>, and <i>TLR6</i>. <i>STAT3</i> expression was repressed by tangeretin and 5-demethylnobiletin, but not by nobiletin. Western blot assay also showed a suppression of expression and phosphorylation of JAK2 by all three PMF monomers, while STAT3 phosphorylation was restrained by tangeretin and 5-demethylnobiletin. The mechanism was primarily verified by the JAK2 inhibitor Ruxolitinib and the STAT3 inhibitor Stattic.