TRAF family member-associated NF-kappa B activator (TANK)-binding kinase1 (TBK1) is a serine- threonine kinase at the crossroads of multiple interferon (IFN)-inducing signaling pathways in innate immunity. The importance of TBK1 in antiviral immunity is well established in mammal models, but in chicken, the molecular characterization and potential function of TBK1 remain unclear. In the present study, the open-reading frame (ORF) of chicken TBK1 (chTBK1) was cloned and characterized. The sequencing results revealed that the chTBK1 ORF consists of 2190 base pairs (bp) encoding a deduced protein of 729 amino acid residues. Multiple sequence alignment analysis demonstrated chTBK1 similarity to other birds and mammals, which indicates that it is evolutionarily conserved. Quantitative real-time PCR (qRT-PCR) results showed that chTBK1 was ubiquitously expressed in chicken tissues and expression was especially high in immune tissues. In addition, the expression of chTBK1 was significantly up-regulated by infection with avian leukosis virus subgroup J (ALV-J) both in vivo and in chicken embryo fibroblasts (CEFs) challenged with ALV-J or stimulated with poly I:C in vitro. Consistent with the activation of chTBK1, the interferon regulatory factor 3 (IRF3) and IFN beta gene in CEFs were also up-regulated after challenge with ALV-J or polyI:C. In contrast, the expression of IRF3 and IFN beta in CEFs was significantly reduced by siRNA targeting the chTBK1 gene compared with a negative control (NC) during ALV-J infection or polyI:C transfection. In conclusion, our results demonstrated that chTBK1 may be an important immunoregulator for IRF3 and IFN beta induction in response to viral stimulation in chicken.