Abstract: Toll-like receptor (TLR), one family of type Ⅰ trans-membrane protein, is a kind of well-studied pattern recognition receptors (PRRs) recognizing pathogen-association molecular patterns (PAMPs). Mammalian TLR3 is best-known for its recognition of pathogen dsRNA and triggering various innate immune responses against virus infection. In contrast to mammalian TLR3, piscine TLR3 can detect not only virus but also bacterial PAMPs. However, whether piscine TLR3 also involved in host anti-parasite immunity is still poorly understood. In the present study, we first cloned an Epinephelus coioides TLR3 (EcTLR3) cDNA sequence by homology cloning and RACE technique. The full length of EcTLR3 was 2937 bp including a 5′-terminal untranslated region (UTR) of 107 bp, a 3′-terminal untranslated region (UTR) of 100 bp and an open reading frame (ORF) of 2730 bp encoding a polypeptide of 909 amino acid residues. The putative isoelectric point (pI) and molecular weight (Mw) of EcTLR3 was 8.27 and 102.68 kD, respectively. The TLR family motifs, such as leucine-rich repeat (LRR) domains and Toll/ interleukin-1 receptor (TIR) domain were also conserved in EcTLR3, with 18 LRR domains and one TIR domain which were connected by one trans-membrane domain. The multiple sequence alignment demonstrated that the full length EcTLR3 amino acid sequence shared high percentage of identities with other teleost (52%—67%), and a somewhat higher sequence identity with the TIR domains (from 55% to 72%), showing that the TIR domain was more highly conserved than that of other domains during TLR3 evolution. To better understand the evolutionary relationship of EcTLR3, a phylogenetic tree was constructed using reported TLR3 protein sequence. The tree indicated that EcTLR3 was branched into the same cluster as that of other teleost TLR3, and showed a highly correlated evolutionary relationship with pufferfish TLR3. To determine the tissues expression pattern of EcTLR3, we examined EcTLR3 expression in healthy grouper by semi-quantitative RT-PCR. The results showed that the EcTLR3 was broadly expressed in all tested tissues, while the expression levels differed noticeably among the tissues, with strong expression in the spleen, trunk kidney and prosencephalon, which imply that EcTLR3 may play an important role in host innate immune response. To identify whether EcTLR3 involved in host anti- Cryptocaryon irritans immune responses, we detected the expression change of EcTLR3 in the skin and spleen at different time points (6h, 1d, 2d, 3d, 5d, 7d, 10d and 14d) post parasite infection by real-time quantitative PCR. The result showed that in the skin, the expression level of EcTLR3 was significantly down-regulated at the early time points (from day 1 to 5), while was up-regulated from day 7 and reached the peak at day 10 post the primary infection. However, in the spleen, the expression level of EcTLR3 was immediately up-regulated and reached the peak at 6h post the parasite infection. The significant up-regulation of TLR3 expression was also observed at day 3 and 5 in the spleen post the primary infection. This result suggested that EcTLR3 may also play an important role in the host defense against C. irritans infection.