Objective Our previous studies have demonstrated that conventional protein kinaseβⅡ (cPKCβⅡ)participated in hypoxic preconditioning (HPC) induced neuroprotection against cerebral ischemic injury.Proteomic analysis revealed that the expression of collapsin response mediated protein (CRMP2) changed significantly in both cytosol and particulate fraction among 49 identified cPKCβⅡ-interacting proteins in cortex of HPC mice.Therefore, this study is to explore the role of cPKCβⅡ-CRMP2 signaling pathway in cerebral ischemic/hypoxic injury and preconditioning of mice.Methods Using our established HPC and middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia mouse models, and the techniques of triphenyltetrazolium chloride (TTC) staining, neurological deficits evaluation and Western blot, we observed the neurological scores, brain infarct volume, the phosphorylation and protein expression levels of CRMP2.Result The phosphorylation level of CRMP2 sharply reduced, while a 55-kDa CRMP2 breakdown product (BDP) increased significantly both in ischemic core and penumbra of ischemic cortex.HPC before ischemia could inhibit both the decrease of pCRMP2 level and increase of BDP production of ischemic cortex.However, cPKCβⅡ inhibitor LY333531 could abolish the HPC-induced inhibitory effect on CRMP2 dephosphorylation and proteolysis.Calpain inhibitor Calpeptin could effectively inhibit the proteolysis of CRMP2 in MCAO treated mice, attenuate the MCAO induced neurological deficits, and decreased brain infarct volume.Conclusion CRMP2 phosphorylation and proteolysis levels were correlated with cPKCβⅡ activation in ischemic cortex of MCAO treated mice.Inhibition of CRMP2 proteolysis could reduce the MCAO-induced ischemic injury.