Toll-like receptor 3 (TLR3) is an important membrane-bound receptor for recognizing double-stranded RNA in innate immunity. In this study, we described the cloning and characterization of the Muscovy duck TLR3 (MdTLR3) gene. The full-length MdTLR3 cDNA (2,836 bp) encoded a polypeptide of 895 amino acids. The deduced amino acid sequence contained 4 main structural domains: a signal peptide, an extracellular leucine rich repeats domain, a transmembrane domain, and a Toll/IL-1 receptor domain. Quantitative real-time PCR analysis indicated that MdTLR3 mRNA was constitutively expressed in all sampled tissues of uninfected Muscovy duck except muscle. Expression of MdTLR3 in brain was significantly upregulated at 24 h (1.94-fold, P < 0.05), reached a peak at 48 h (4.64-fold, P < 0.05), and recovered to normal levels at 72 h postinfection with the H5N1 highly pathogenic avian influenza virus. In contrast, MdTLR3 expression was downregulated during the test period in spleen and lung. These results implicated MdTLR3 was a novel member of the TLR family, which is involved in the early stage of antiviral innate immunity.