Chromatographic fractionation of Sigesbeckia glabrescens led to the identification of 10 new sesquiterpene lactones, named siegesbeckialides I-O (1-7) and glabrescones A-C (8-10), along with 14 known analogues. An anti-inflammatory activity assay showed that siegesbeckialide I (1) most potently inhibited LPS-induced NO production in RAW264.7 murine macrophages. Furthermore, siegesbeckialide I suppressed the protein expression of iNOS and COX2, as well as the release of PGE2, IL-1β, IL-6, and TNF-α in LPS-stimulated RAW264.7 cells. Mechanistically, siegesbeckialide I directly binds to inhibitors of IKKα/β and suppresses their phosphorylation. This leads to the inhibition of IKKα/β-mediated phosphorylation and degradation of inhibitor α of NF-κB (IκBα), as well as the activation of NF-κB signaling.