Endotoxin, also known as lipopolysaccharide (LPS), is the major mediator of septic shock due to Gram-negative bacterial infections.Our previous study found that CLP-19, a looped peptide derived from limulus anti-lipopolysaccharide peptide with high LPS-neutralizing activity,have showed immunoregulatory effect but the molecular mechanism is unclear.In this study, Our results shows that CLP-19 significantly increased the survival rate in lethal LPS shock mouse models, decreased the serum TNF-α level and reduced the pathological injury of lung 20 h prior to LPS challenge.CLP-19 also induced endotoxin tolerance (ET) phenotype in RAW 264.7 cells in vitro in a dose-dependent and time-dependent manner, which attenuated LPS-induced TNF-α and IL-6 secretion and increased anti-inflammatory factor, IL-10 production.Pretreated by CLP-19,the expressions of Toll-like receptors 4 (TLR4) on the cell surface remarkably decreased by LPS re-stimulatation.Moreover, CLP-19 pretreatment inhibited degradation of inhibitor of nuclear factor (NF)-κB (I-κB) rather than influenced expression of myeloid differentation 88 (MyD88) and TIR domain-containing adapter inducing IFNγ 6 (TRIF6), resulting in decreased expression of inflammatory cytokine production.Taken together with the finding suggested that CLP-19 induces ET phenotype via inhibiting NF-κB activation.In conclusion, we have revealed a novel function of CLP-19 that appears to represent a potential therapeutic agent for clinical treatment of sepsis.