Abstract To investigate the effect of chronic ketamine injection on expression of c-Fos and pCREB in Kunming mice retrosplenial cortex. Methods: The adult male Kunming mice were divided into four groups randomly: the sham group, 50 mg/kg ketamine group (K1 group), 100 mg/kg group (K2 group), 200 mg/kg group (K3 group), intraperitoneal injected ketamine or normal saline at 5-day intervals for successively 6 times. The mice were sacrificed and fixed with 4% paraformaldehyde. Their brains were removed for frozen-sectioning. The expression of c-Fos and pCREB were detected with immunohistochemistry. Results: (1) There is a basal expression of c-Fos and pCREB in Retrosplenial cortex. (2) Chronic ketamine injection inhibited the expression of c-Fos in Retrosplenial cortex in a dose-dependent manner; meanwhile, the expression of pCREB was increased in retrosplenial in a dose-dependent manner. Conclusion: Chronic ketamine injection inhibit the expression of c-Fos expressions in retrosplenial cortex, the pCREB may involved the inhibit effect of ketamine.