Toll-like Receptor (TLR) signaling cascade proceed with the help of several co-receptors and adaptor proteins resulting in the activation of a protective immune response against the invading pathogens. TLR signaling pathway can be activated through the recruitment of either Myeloid Differentiation factor 88(MyD88) or Toll-Interleukin-1 receptor (TIR)-domain-containing adapter-inducing interferon-β (TRIF). TLR4, on the other hand, is endowed with the ability of activating both. During the recognition of Lipopolysaccharide (LPS) by TLR4, LPS binding protein (LBP) detaches the monomeric LPS and transfer it to CD14, which in turn exposes the captured LPS to the TLR4/MD2 complex, leading to the subsequent activation of the downstream signaling cascade and the secretion of pro-inflammatory cytokines. However, a dysregulated TLR4 response is often a hallmark of various inflammatory and autoimmune diseases, for this reason, TLRs are being actively pursued as drug targets in the treatment of immune-related disorders. Herein, we report a TIRAP-derived decoy peptide, Ajou5, which displays significant inhibitory effect on TLR4-mediated pathway. In RAW267.4 and THP-1 cells, Ajou5 inhibits the NF-κB activation monitored by SEAP assay, hinders the LPS-induced secretion of inflammatory cytokines such as TNF-α and IL-6, and inhibits the activation of Mitogen-activated protein kinases (MAPKs) and Interferon regulatory factor 3 (IRF3). Ajou5 was unable to inhibit inflammatory cytokines induced by other TLR ligands. In silico data suggested that the interaction of LBP and CD14 is similar to the interaction between Ajou5 and CD14 suggesting that Ajou5 can interrupt ligand transfer to CD14 by delaying the CD14-mediated signaling pathway.