Abstract: Toll like receptor 21 (TLR21) is a non-mammalian Toll-like receptor that recognizes unmethylated CpG DNA to trigger innate immune responses against pathogenic microorganism infection. The present study cloned the full-length cDNA sequence of TLR21 (AjTLR21) from Japanese eel, Anguilla japonica, which had the common characteristics of Toll like receptors (TLRs). The intronless AjTLR21 shared similar gene structure with other vertebrate TLR21 genes. AjTLR21 widely expressed in all examined tissues/organs with the highest expression in blood. Poly I:C treatment significantly induced the expression of AjTLR21 in spleen and kidney at 8 hours and in blood, gill, intestine and spleen at 16 hours (P < 0.05). Luciferase reporter assay exhibited that the 5' flanking region of the AjTLR21 contains positive regulatory elements for Poly I:C induced AjTLR21 expression. The stimulation of Edwardsiella tarda also increased the expression of AjTLR21 in blood and kidney at both 16h and 72h, suggesting an important role in anti-bacterium immune response. Our results revealed multiple effects of teleosts TLR21 in immune system, which has impor-tant theoretical significance and application value to understand the immunological function of AjTLR21.