As an important adaptor protein in the intracellular signaling of interleukin-1 receptor/toll-like receptor （IL-1R/TLR） superfamily，myeloid differentiation factor 88 （MyD88） plays a crucial role in the innate immune response. So far，no report about MyD88 in half smooth tongue sole （Cynoglossus semilaevis），one of the most promising and commercially important species for marine fish culture in China，is available. In this paper，the cloning，characterization and expression pattern of MyD88 in half smooth tongue sole was described. The full length MyD88 cDNA of half smooth tongue sole is 1 612 bp including a 132 bp 5 ′ untranslated region （UTR），a 590 bp 3 ′ UTR and a 858 bp open reading frame （ORF） encoding 285 amino acids. MyD88 contains a typical death domain at the N-terminal and a conservative Toll/IL-1R （TIR） domain at the C-terminal. The length of genomic DNA of MyD88 in this species is 2 923 bp with 5 exons and 4 introns. The phylogenetic tree based on 11 species MyD88 proteins showed that half smooth tongue sole MyD88 shared the closetest relationship with Japanese flounder （Paralichthys olivaceus）. The next quantitative real-time reverse transcription PCR analysis revealed MyD88 gene was expressed in all tested tissues with higher expression in immune tissues and reproduction tissues including blood， liver，spleen，kidney，testis and ovary. Then，the stimulation with LPS （lipopolysaccharide），GPN （peptidoglycan） and poly I：C showed that these stimulators could induce up-regulation of MyD88 expression in PBL of half smooth tongue sole. Finally，the pathogen infection experiment with Gram negative bacteria Vibrio anguillarum，indicated that MyD88 expression was significantly increased in the spleen by nealy 180 times and in the liver by more than 60 times after 96 th hour and in the headkidney with near 5 times at 48 th hour after challenge，respectively. These results suggest that MyD88 play an important role in the innate immune system in half smooth tongue sole.