Abstract: Objective To explore the role of Calcium/calmodulin-dependent protein kinaseⅡ(CaMKⅡ) in the development of cerebral hypoxic preconditioning(HPC). Methods Healthy male BALB/c mice (weighting 18～22g, 8～10w) were randomly divided into 7 groups as follows: normoxic control (H0), early(H1-H4)and delayed (H5 and H6) hypoxic preconditioned mice groups. SDS-PAGE, Western blot and Gel Doc imagine systems were applied to quantitatively analyze the level of CaMKⅡ phosphorylation and protein expression level in the brain of mice. Results Compared with H0 group, the phosphorylation level of CaMKⅡ increased in cortex and hippocampus of mice in H3-H5 hypoxic preconditioned groups(P<0.05, n=6). However, there was no significant changes in total CaMKⅡprotein expression in cortex and hippocampus of hypoxic preconditioned mice. Similarly, enhanced p-Thr286 CaMKⅡwas also observed in the hippocampus and cortex of mice by immunostaining following hypoxic exposures (H3 and H6). Conclusion The increased phosphorylation of CaMKⅡ may involved in the development of cerebral HPC in mice.