Inflammation can be initiated by invading microbial pathogens. Toll-like receptors (TLRs) recognize molecular structures derived from microbial pathogens and regulate the activation of innate immunity. In general, TLRs have 2 major downstream signaling pathways, the myeloid differentiation primary response protein 88 (MyD88)- and Toll/interleukin 1 receptor (TIR) domain-containing adaptor protein (TIRAP) inducing interferon ${eta}$ (TRIF)-dependent pathways, which lead to the activation of nuclear factor (NF)-${kappa}B$ and interferon regulatory factor 3 (IRF3). Costunolide, one of the active ingredients in Aucklandiae Radix (Saussurea lappa), has been used to treat many chronic inflammatory diseases. To evaluate the therapeutic potential of costunolide, its effect on signal transduction via the TLR signaling pathways was examined. Costunolide inhibited lipopolysaccharide or polyinosinic-polycytidylic acid-induced NF-${kappa}B$ and IRF3 activation and IRF3 phosphorylation, as well as interferon-inducible genes such as interferon inducible protein-10. The results suggest that costunolide can modulate immune responses regulated by TLR signaling pathways and may be the basis of effective therapeutics for chronic inflammatory diseases.