Background: Ophiopogon japonicas (L.f) Ker-Gawl has been used as a traditional Chinese medicine to cure acute and chronic inflammation and cardiovascular diseases including thrombotic diseases for thousands of years. Previous phytochemical studies showed that O. japonicus contained compounds with anti-inflammatory activity. The aim of this study was to identify and isolate compounds with anti-inflammatory activity from the rhizome of O. japonicas. Methods: Compounds were isolated by various column chromatography and their structures were identified in terms of nuclear magnetic resonance spectrum (NMR) and mass spectrum (MS). To measure the anti-inflammatory effects of thirteen compounds in LPS-induced RAW 264.7 macrophage cells, we used the following methods: cell viability assay, nitric oxide assay, enzyme-linked immunosorbent assay, quantitative real-time PCR analysis and western blotting analysis. Results: One new and twelve known compounds (mainly homoisoflavonoids) were extracted from O. japonicas, in which 4'-O-Demethylophiopogonanone E (10) was considered as a new compound, additionally, compounds 4-O-(2-Hydroxy-1-hydroxymethylethyl)-dihydroconiferyl alcohol (2) and 5,7-dihydroxy-6-methyl-3-(2', 4'-dihydroxybenzyl) chroman-4-one (12) were isolated from the rhizome of O. japonicas for the first time. The isolated compounds Oleic acid (3), Palmitic acid (4), desmethylisoophiopogonone B [5,7-dihydroxy-3-(4'-hydroxybenzyl)-8-methyl-chromone] (5), 5,7-dihydroxy6- methyl-3-(4'-hydroxybenzyl) chromone (7) and 10 significantly suppressed the production of NO in LPS-induced RAW 264. 7 cells. Especially compound 10 showed the strongest effect against the production of the pro-inflammatory cytokine IL-1 beta and IL-6 with the IC50 value of 32.5 +/- 3.5 mu g/mL and 13.4 +/- 2.3 mu g/mL, respectively. Further analysis elucidated that the anti-inflammatory activity of compound 10 might be exerted through inhibiting the phosphorylation of ERK1/2 and JNK in MAPK signaling pathways to decrease NO and pro-inflammatory cytokines production. Conclusions: Our results indicated that 4'-O-Demethylophiopogonanone E can be considered as a potential source of therapeutic medicine for inflammatory diseases.