We recently reported that nucleotidebinding oligomerization domain (NOD)2, an important cytoplasmic pattern recognition receptor, is involved in cerebral ischemiareperfusion (I/R) injury.βarrestins, in addition to regulate desensitization of G proteincoupled receptors (GPCRs), have emerged as potential mediators of innate immune activation.However, the role and mechanism of βarrestin2 in NOD2triggered signaling in the cerebral I/R remain to be established.Methods BV2 cells were transfected with either βarrestin2shRNA plasmid or βarrestin2 fulllength plasmid and control vector.Middle cerebral artery occlusion (MCAO) was induced in male wildtype mice and in wild type (WT) and βarrestin2 deficient mice.Results muramyl dipeptide (MDP), an extrinsic ligand of NOD2, significantly increased the expression of TRAF6 and COX2 and enhanced the activation of NFκB in the microglia timedependently.MDP stimulation also promoted the expression and activation of MMP9 timedependendy, but did not affect MMP2 obviously.Additionally, βarrestin 2 interacted with TRAF6 after MDP stimulation rapidly.Overexpression of βarrestin2 inhibited NFκB and MMP9 activation and COX2 upregulation induced by MDP, while silence of βarrestin2 enhanced NOD2triggered inflammatory signaling.Finally, Deletion ofβarrestin 2 markedly aggravated brain infarction, neurological deficit and inflammation induced by MDP in mice subjected to MCAO.Conclusion The results provide the first evidence that 3arrestin 2 is an essential negatively regulator of NOD2 triggered inflammatory signaling in the cerebral I/R injury.