Abstract：Objective To investigate the mechanism of MyD88 inhibition of hepatitis B virus (HBV) replication. Methods Two truncated versions of MyD88, M (1-151) and M (152-296) were constructed. Plasmid IκBα-SR encoding NF-κB super-repressor or plasmid IKKα/IKKβ which can strongly activate NF-κB signaling were used. Huh7 cells were transiently transfected with HBV dimer, MyD88 or the above plasmids. Assays of HBsAg, HBeAg, core protein, HBV DNA and NF-κB activation were performed. Results The expression of the full length MyD88, and the two truncated forms, M (1-151) and M (152-296), showed different induction patterns of NF-κB activity; patterns that are consistent with their inhibitory effect on viral protein synthesis and core particle-associated HBV DNA replication. Similarly, the expression of MyD88 resulted in a significant reduction of core protein and the co-expression of IKKα/IKKβ dramatically inhibited the core protein level. Furthermore, the co-expression of IκBα-SR dramatically restored the core protein level. Similar results were also observed for HBeAg, HBsAg and HBV core particle DNA. Conclusion Results from these studies support a role of NF-κB signaling activation in HBV viral replication and suggest a novel mechanism for the inhibition of HBV replication by MyD88.