Interleukin-26 (IL-26) belongs to the IL-10 cytokine family and exerts diverse biological functions in regulating immune responses in vertebrates. Although IL-26 has been extensively studied in mammals, the functions of IL-26 remain largely unexplored in lower vertebrates. In this study, we determined the tissue and cell sources of IL-26 using a monoclonal antibody (mAb) generated against grass carp (Ctenopharyngodon idella, Ci) IL-26, and investigated the responses of IL-26 producing cells to bacterial infection. We showed that the CiIL-26 mAb specifically recognized the recombinant CiIL-26 proteins expressed in the Escherichia coli and HEK293 cells. Flow cytometry analysis revealed that the CiIL-26 mAb could detect the intracellular CiIL-26 expressed in the HEK293 cells and CIK cells stimulated with inactivated Aeromonas hydrophila (A. hydrophila). Using confocal microscopy, we analyzed IL-26