Toll-like receptors play important roles in detecting pathogen-associated molecular patterns for initiation of immune responses against microbial infections. Toll-like receptor 8 (TLR8) belongs to a subfamily comprising TLR7, TLR8 and TLR9, and recognizes viral single-stranded RNA and small molecular weight agonists to activate anti-viral immune responses. TLR8s from different species have distinct ligand recognitions. TLR8s from several non-rodent species including cat, horse, sheep, and bovine are activated by small molecular TLR8 agonists, whereas TLR8s from mouse and rat, two rodent species, are activated only in the presence of PolyT-ODN. The lack of a five-amino-acid motif in the undefined region following the leucine-rich repeat 14 of the mouse and rat TLR8 ectodomains has been suggested to be a reason for the weak activities of these two rodent TLR8s. To further investigate the activation of TLR8, in this study we cloned rabbit TLR8 (rabTLR8) cDNA. The rabTLR8 has a longer undefined region in its ectodomain than the TLR8 from other species. In cell-based assay, this rabTLR8 is activated by TLR8 ligand in the absence of PolyT-ODN. Nevertheless, upon stimulation the rabTLR8 had a lower activation compared to the activation of TLR8 from other species, except the mouse and rat TLR8s. Using different deletion and human-rabbit chimeric TLR8 expressing constructs, we showed that the extra peptide in the undefined region played a role in determining the activity of rabTLR8. These results suggest that the undefined region plays a role in determining the activity of rabTLR8.