Abstract: In order to establish a stable eel cell line conducive to studying eel immune response, this study utilized the tissue block method to isolate and cultivate a heart cell line from Japanese eel (Anguilla japonica). Designated as AJH (Anguilla japonica Heart cells, AJH), these cells were cultured in DMEM/F-12 medium containing 20% fetal bovine serum (FBS) at 28℃, with passages conducted every 3 to 4 days. Subsequent to the 5th generation, cells were maintained in DMEM/F-12 medium with 15% FBS for ongoing passage culture. Ribosomal RNA analysis confirmed the Japanese eel origin of the cells. Karyotype analysis revealed that AJH cells possessed 38 chromosomes, comprising 10 metacentric, 10submetacentric, and 18 acrocentric chromosomes. Viral infection experiments showed efficient propagation of Anguilla herpesvirus 1 (AngHV-1) within AJH cells. EGFP-AjLC3 transfection into AJH cells was carried out using a variety of reagents, and transfection efficiency was detected by flow cytometry, with the highest observed transfection efficiency in AJH cells reaching (37.17±0.12)%. In addition, co-transfection of AjIRF1 with AjIFNa or AjIFNc1 promoters into AJH cells resulted in up-regulation of AjIFNa or AjIFNc1 promoter activities, indicating the potential utility of AJH cells in gene function studies. After LPS stimulation, expression levels of AjTNF-α and AjIL-Iβ in AJH cells were significantly up-regulated. Similarly, after Poly I:C transfection, expression levels of AjRIG-Ia and AjRIG-Ib were significantly up-regulated, suggesting the inducible nature of immune responses in AJH cells by LPS and Poly I:C. Overall, the establishment of the Japanese eel heart cell line in this study presents a valuable cellular model for elucidating the mechanisms underlying antiviral and antibacterial immune responses in eels.