MyD88 is a critical adaptor in the TLRs signaling pathway, which can activate NF-KB signaling pathway and promote proinflammatory cytokines production. However, the molecular mechanisms that modulate MyD88 expression, especially in teleost, remain largely unknown. In this study, we showed that NOP56 serve as a negative regulator of the MyD88-mediated NF-KB signaling pathway. NOP56 overexpression inhibited the protein expression of MyD88. Whereas, siRNA knockdown of NOP56 had opposite effect. Furthermore, we found that the NOSIC domain is responsible for the suppressive effect of NOP56 in MyD88 expression at protein level. Therefore, we identified NOP56 as a negative regulator of MyD88-mediated NF-KB signaling by inhibiting MyD88 expression and provided new insight into the regulation mechanism in teleost fish.