Innate immune responses are crucial for the host to effectively clear pathogens and repair damaged tissues, but their dysregulation of has been associated with numerous illnesses, such as cancer, sepsis, metabolic disorders and chronic inflammatory diseases. Therefore, the innate immune responses must be tightly regulated. Toll-like receptors (TLRs) are major components of the innate immune system. Different mechanisms have been demonstrated to regulate TLRs-driven immune responses, including post-transcriptional regulation. Post-transcriptional regulation is a flexible but specific mechanism to modulate the expression of inflammatory-related genes. However, the molecular mechanism of post-transcriptional regulation still remains indistinct. We recently identified a novel terminal uridyltransferase (TUTase), which was induced by TLR ligands treatment. TUTases are known to have the ability to uridylate or adenylate small RNAs and mRNAs, thereby modulating the stability of the target RNAs. Nevertheless, the function of this novel TUTase in TLR4-mediated immune response remains to be discovered. We demonstrated that depletion of this TUTase in RAW 264.7 macrophages led to dysregulation of several cytokines production including IL-6 upon LPS challenge. Down-regulation of IL-6 mRNA was also observed in TUTase-deleted bone marrow-derived macrophages after stimulation of TLR4 agonist. We further demonstrated that this TUTase regulated IL-6 mRNA stability after LPS stimulation through the control of Regnase-1 mRNA uridylation and degradation. Taken together, our findings reveal the functional role of the novel TUTase in TLR4-mediated immune responses by modulating the mRNA stability of certain inflammatory mediators.