ABSTRACTAs a member of the pattern recognition receptors (PRRs) involving in the innate immune system, Toll-like receptors (TLRs) can sense a wide range of microbial pathogens and combat infections by producing antimicrobial products, inflammatory cytokines, and chemokines. All TLRs, with the exception of TLR3, activate a signalling cascade via the myeloid differentiation primary response gene 88 (MyD88). Therefore, the activation of MyD88-dependent signalling pathway must be finely controlled. Herein, we identified that cyclin-dependent kinase 5 (CDK5) negatively regulated TLR-MyD88 signalling pathway by targeting MyD88. Overexpression of CDK5 reduced the production of interferons (IFNs), while a deficiency in CDK5 increased the expression of IFNs in response tovesicular stomatitis virus(VSV) infection. Mechanistically, CDK5 suppressed the formation of MyD88 homodimers, resulting in the attenuated production of IFNs induced by VSV infection. Surprisingly, its kinase activity does not play a role in this process. Therefore, CDK5 can act as an internal regulator to prevent excessive production of IFNs by restricting TLR-MyD88-induced activation of antiviral innate immunity in A549 cells.