The regulation and mechanism of action of the avianspecific Toll-like receptors (TLR) genes have been characterized in chicken macrophages in the present study. Chicken TLR15 is a novel pattern recognition receptor whose cognate ligand is unknown and is structurally different from other TLRs. Screening various stimulants in macrophage cell cultures, we concluded that TLR15, in collaboration with TLR2 and TLR21, is involved in CpG oligonucleotide (ODN) response in chicken macrophages. We further verified CpG responsiveness by using inhibitor ODN in macrophage cells. Additionally, we determined the pathway use of MyD88 adaptor molecule after stimulation with CpG-ODNs, resulting in IL1B expression. The current study expands the possibilities for vaccine development studies by discovering disease response pathways.