IL-20 is a pleiotropic cytokine that belongs to the IL-10 family and plays an important biological role in tissue homeostasis and regulation of host immune defenses. IL-20 homologues have recently been discovered in fish, but their functions have not been studied. In this study, an IL-20 like (IL-20L) cytokine was cloned in grass carp (Ctenopharyngodon idella) and its bioactivities were investigated. Expression analysis showed that the Ci IL-20L gene was constitutively expressed in tissues with the highest expression detected in the head kidney. It was upregulated in the head kidney after infection with Flavobactrium columnare (F. cloumnare) and grass carp reovirus II (GCRV II). The recombinant Ci IL-20L produced in E. coli cells was shown to be effective in inducing the expression of Th cytokine genes (IFN-γ, IL-4/13A, IL-4/13B and IL-10), macrophage marker genes (arginase 2, IRF4, KLF4 and SOCS3) and inflammatory genes (IL-1β, IL-6, IL-8 and TNFα) in the head kidney leukocytes when stimulated at 12 h. Long term culture (6 days) of head kidney macrophages in the presence of Ci IL-20L leads to high expression of IRF4, TGFβ1 and arginase 2. Our data suggest that IL-20 may play regulatory roles in promoting Th responses, macrophage differentiation and inflammation. • An IL-20 like (IL-20L) cytokine was characterized in grass carp. • Ci IL-20L was upregulated during infection with Flavobactrium columnare and grass carp reovirus II. • Ci IL-20L transiently induced the expression of genes associated with Th response and inflammation. • Long term exposure of Ci IL-20L promotes M2 macrophage polarization in vitro. [ABSTRACT FROM AUTHOR]