Amentoflavone is naturally occurring bioflavonoid that is found in a number of plants. In this paper, the anti-inflammatory activity of amentoflavone in LPS-stimulated macrophages and its mode of action were examined. Using LPS-stimulated RAW264.7 macrophage cells, we found that amentoflavone exerted anti-inflammatory activities through inhibition of nitric oxide (NO) production and tumor necrosis factor (TNF)-${alpha}$ and macrophage inflammatory protein (MIP)-2 secretion. Amentoflavone (1.0-20 ${mu}M$) gradually inhibited nitrite production without cytotoxicity. Amentoflavone (1.0 and 10 ${mu}M$) effectively suppressed both TNF-${alpha}$ and MIP-2 cytokine release from LPS-stimulated RAW264.7 cells. The expression of mIL-$1{eta}$ and mMIP-2 cytokine mRNAs was completely inhibited while expression of mMIP-1 was effectively suppressed and mTNF-${alpha}$ expression was slightly inhibited by 10 ${mu}M$ amentoflavone. We also demonstrated that the innate immune response to amentoflavone involves the toll-like receptor (TLR) and mitogen-activated protein kinase (MAPK) pathways. LPS-induced upregulation of p38 MAPK phosphorylation was significantly reduced by 10 ${mu}M$ amentoflavone. These results suggest that amentoflavone exhibits effective anti-inflammatory activities through regulation of TLR4 and phosphorylation of p38 MAPKs.