Abstract Background This study aims to identify the major anti-inflammatory components in the petroleum ether extract of Bupleurum malconense (Chaihu), by bioassay-guided fractionation, and to investigate the anti-inflammatory mechanisms of active components in lipopolysaccharide (LPS)-stimulated murine macrophage RAW-Blue cells. Methods A QUANTI-Blue assay was used to guide fractionation of B. malconense root extract. The petroleum ether extract which exerted significant secreted embryonic alkaline phosphatase (SEAP) inhibition effect was purified by silica gel column chromatography and assisted with reverse phase HPLC. The major bioactive compound which significantly inhibited SEAP activity was obtained and its anti-inflammatory effects in LPS-induced RAW-Blue cells were measured by the overproduction of NO (Griess method), gene expression of Il-1β, Tnf-α and iNos (real-time PCR). In parallel, protein expressions of COX-2, iNOS and IκB-α were determined by western blot. Results In bioassay-guided fractionation using LPS-stimulated mouse macrophage RAW-Blue cells, (+)-3′-angeloxyloxy-4′-keto-3′,4′-dihydroseselin (Pd-Ib) was identified by MS and NMR spectral analyses. Pd-Ib (5, 10, 20 μg/mL) suppressed the gene expression of Il-1β (P