Toll-like receptors (TLRs) play an important role in induction of innate immune responses. TLRs can trigger the activation of myeloid differential factor 88 (MyD88)- and Toll-interleukin-1 receptor domain-containing adapter inducing interferon-beta (TRIF)-dependent downstream signaling pathways. Expression of more than 70% of lipopolysaccharide (LPS)-induced target genes is mediated through a TRIF-dependent signaling pathway. To evaluate the therapeutic potential of isoliquiritigenin (ILG), we examined its effect on signal transduction via the TRIF-dependent pathway of TLRs. ILG inhibited interferon regulatory factor 3 activation induced by LPS or polyinosinic-polycytidylic acid, as well as interferon-inducible genes, such as interferon-inducible protein-10. ILG attenuated ligand-independent activation of IRF3 induced by TRIF or TBK1. Furthermore, ILG inhibited TBK1 kinase activity in vitro. Together, these results demonstrate that TBK1 is the molecular target of ILG, resulting in the downregulation of the TRIF-dependent signaling pathways of TLRs.