Abstract: Myeloid differentiation factor 88 (MYD88) is a key adapter protein in Toll-like receptor (TLR) signaling, which plays significant role on the innate and adaptive immunity. In this study, a myd88 gene from lamprey (Lampetra japonica) was obtained. The ORF of myd88 was 852 bp in length, encoding a polypeptide of 283 amino acids. The theoretical molecular weight of lamprey Myd88 was 32.432 kD with a theoretical isoelectric point of 6.25. Lamprey Myd88 was predicted to have no signal peptide. Multiple protein sequence alignment revealed highly conserved death domain in N-terminal, and Box1, Box2, and Box3 in the C-terminal TIR domain, which indicated that lamprey Myd88 was homologous with the MYD88 in other species. Quantitative real-time PCR analysis showed lamprey myd88 gene was extensively expressed in all detected tissues. The highest expression level was observed in gill, which was followed by marrow and kidney. Under in vivo stimulation by lipopolysaccharide (LPS), the expression of myd88 significantly increased in leukocytes, followed by gill, which implicated its role in the defense of L. Japonica against bacteria. Furthermore, increasing expressions of downstream proteins in Myd88-dependent TLR signaling pathway, including Irak1, Traf6, Ikkβ, and Nfkb, were detected in the tested tissues stimulated with LPS. These results suggested that a conserved Myd88-dependent TLR signaling pathway was found in the lamprey, which play a fundamental role on the exploration of the origin and evolution of the signaling pathway in immune response in future.