Nuclear factor (NF)-κB regulates a central common signaling for immunity and cell survival. Artemisolide (ATM) was previously isolated as a NF-κB inhibitor from a plant of Artemisia asiatica. However, molecular basis of ATM on NF-κB activation remains to be defined. Here, we demonstrate that ATM is a typical inhibitor of IκB kinase β (IKKβ), resulting in inhibition of lipopolysaccharide (LPS)-induced NF-κB activation in RAW 264.7 macrophages. ATM inhibited the kinase activity of highly purified IKKβ and also LPS-induced IKK activity in the cells. Moreover, the effect of ATM on IKKβ activity was completely abolished by substitution of Cys-179 residue of IKKβ to Ala residue, indicating direct targeting site of ATM. ATM could inhibit IκBα phosphorylation in LPS-activated RAW 264.7 cells and subsequently prevent NF-κB activation. Further, we demonstrate that ATM down-regulates NF-κB-dependent TNF-α expression. Taken together, this study provides a pharmacological potential of ATM in NF-κB-dependent inflammatory disorders.