As an important serine/threonine kinase in the IKK family, TANK Binding Kinase 1 (TBK1) plays a critical role in innate immune response by activating NF-κB and type I IFN signaling pathways in mammals. Although several studies have reported that fish TBK1 was involved in the regulation of type I IFN production, information on TBK1's close association with antimicrobial immune responses in the regulation of NF-κB and MAPK signaling pathways is still limited in teleost fish. In order to elucidate the regulation of fish TBK1 in NF-κB, I IFN, and MAPK immune response signaling pathways, the full-length cDNA of a TBK1 homologue, AjTBK1, was cloned by SMART RACE from Japanese eel, and its characteristics of expression in response to various PAMPs and Aeromonas hydrophila infection were investigated both in vivo and in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). In addition, the subcellular localization of AjTBK1 GFP fusion protein and the induction of AjTBK1 overexpression in the activation of NF-κB, AP1 and type I IFN performed by Dual-Glo luciferase assay system were also detected. Amino acid sequence analysis indicated that AjTBK1 encodes a polypeptide of 731 amino acids, which has the conserved N-terminal kinase domain (KD), a ubiquitin-like domain (ULD), a scaffold dimerization domain (SDD), and a C-terminal domain (CTD). The predicted three-dimensional structure of AjTBK1 is similar to that of human TBK1, and AjTBK1 is clustered with other fish families in the phylogenetic tree. Quantitative real-time PCR (qRT-PCR) analysis revealed that AjTBK1 is broadly expressed in a wide range of tissues, with high expression in liver and intestine. In vivo, the expression of AjTBK1 in the liver of Japanese eel was significantly increased by 1.8- fold at 6h, 1.8-fold at 6 h, and 1.9-fold at 48 h after injection with LPS, viral mimic poly I:C and A. hydrophila infection, respectively. The AjTBK1 expression in kidney was found to increase at 12 h and 24 h with 1.9- and 1.6-fold after A. hydrophila infection, but decreased following the stimulation of LPS and poly I:C. In vitro, the AjTBK1 transcripts of Japanese eel liver cells were significantly enhanced to its peak by the treatment of LPS, poly I:C, PGN, or the 10⁶ CFU/mL A. hydrophila, being up to the 15.3-, 5.8-, 9.3- and 4.7-fold, respectively. Subcellular localization studies showed that AjTBK1 was evenly distributed in the cytoplasm of HEK293 cells in natural state. AjTBK1 was found to aggregate into spots in the cytoplasm upon the stimulation of LPS or poly I:C in HEK293 cells. Additionally, luciferase assays demonstrated that the AjTBK1 overexpression significantly enhanced the activation of NF-κB, AP1, and IFNβ-responsive promoters in HEK293 cells, and robustly up-regulated the activation of NF-κB, AP1, and IFNβ-responsive promoter, which were 1.8-, 1.2-, and 1.92-fold induced at 24 h, 12 h, and 24 h after cotransfection, respectively. These results collectively suggest that AjTBK1 may function as important positive regulation in innate immunity of host against antibacterial and antiviral infection likely via the activation of NF-κB, AP1, and type I IFN signaling pathways.