Morus alba L. bark has been widely used in traditional medicine for treating several inflammatory diseases, such as hypertension, diabetes mellitus and coughing, however, the molecular mechanisms of its anti-inflammatory effect has been not clearly understood. In this study, we examined the effect of an extract of Morus alba L. bark (MabE) on TLR-induced activation of RAW264.7 macrophages. RAW264.7 cells were stimulated with polyI:C (TLR3 ligand), lipopolysaccharide (LPS, TLR4 ligand), or Imiquimod (IMQ, TLR7 ligand) with or without MabE and measured NF-kB activation and cytokine production. By using luciferase reporter assay, we found that MabE inhibited the activation of NF-kB. In addition, MabE inhibited the phosphorylation of p65, extracellular-signal-regulated kinases (ERK), and p38 in RAW264.7 cells. In accordance with the inhibition of inflammatory signals, MabE also inhibited the production of IL-6 and IL-1 from TLR ligands-treated RAW264.7 cells. These results suggest that the anti-inflammatory effect of MabE can be mediated by the inhibition of NF-kB /p38 /ERK inflammatory pathways. Collectively, these results indicate that MabE and their active compounds are promising targets for developing novel anti-inflammatory drug.