In order to clone the peafowl (Pavo cristatus) Toll-like receptor 7 (TLR7) gene and study its biological function, the peafowl TLR7 coding sequences (CDS) were amplified by PCR of cDNA from the whole spleen of peafowl. The full-length sequence of the peafowl TLR7 gene CDS is 3,141 bp and encodes a 1,046-amino acid protein with a classic TLR composition of 16 leucine-rich repeats (LRR). Insertions of amino acids were found at position 15 of LRR2, LRR5, LRR7, LRR9, LRR11, LRR12, LRR14, and LRR15; and position 10 of LRR11. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that the peafowl TLR7 gene was highly expressed in lymphoid tissues of the spleen, bursa, bone marrow, lung, and peripheral blood mononuclear cells (PBMC). HEK293T cells were transfected with a peafowl TLR7 plasmid, and functional analysis showed that peafowl TLR7 could respond to R848, leading to activation of NF-κB. Following R848 stimulation or Newcastle disease virus infection of peafowl PBMC, the levels of IL-1β, IFN-γ, CCLi2, and TGF-β4 mRNA, assessed by quantitative real-time PCR, increased significantly. Triggering peafowl TLR7 results in upregulation of inflammatory cytokines and chemokines, suggesting that peafowl TLR7 plays an important role in the innate immune response.