Objective To investigate the effects of naloxone injected into cisterna magna on S100β protein and neuron-specific enolase (NSE) levels in serum and the histopathology of brain tissue of rats following cardiopulmonary resuscitation(CPR). Methods Thirty adult male SD rats were randomly divided into 3 groups: Sham group, Conventional CPR group (intravenous injection of epinephrine, 0.2 mg/kg) and Naloxone CPR group(cisterna magna injection of naloxone, 2 mg/kg). Asphyxiation was used to set up rat cardiac arrest model, and corresponding drugs were given when the resuscitation was carried out. The blood samples were taken from orbital venous plexus at 0.5 h, 3 h, 6 h and 24 h after restoration of spontaneous circulation (ROSC). Enzyme-linked immunosorbent assay (ELISA) was used to detect serum levels of S100β protein and NSE. Brain tissue was taken after the last blood sampling and the pathology of brain was observed by hematoxylin-eosin (H-E) staining. Results Serum S100β protein levels of the Conventional CPR group and Naloxone CPR group were significantly higher than those of the Sham group at all time points (P <0.01); compared with the Conventional CPR group, S100β protein levels in Naloxone CPR group were significantly decreased at 3 h, 6 h, and 24 h after ROSC (P <0.05 or P <0.01). Serum NSE protein level of the Conventional CPR group at all time points and Naloxone CPR group at 6 h and 24 h after ROSC were significantly higher than those of the Sham group (P <0.05 or P <0.01). Serum NSE protein levels were significantly decreased at 6 h and 24 h after ROSC in Naloxone CPR group compared with the Conventional CPR group (P <0.05 or P <0.01). Moreover, hippocampus glial cells of Conventional CPR group were scattered and decreased, with condensed eosinophilic cytoplasm, narrowed nuclues, unclear nucleolus, and swollen and deformed capillaries. However, most nerve cells of Naloxone CPR group had rich cytoplasm and the nucleolus was clear; only a few nerve cells and capillaries showed edema-like changes of different degrees. Conclusion Naloxone injected into cisterna magna has a prominent protective effect on the brain of rats following cardiopulmonary resuscitation.