Objective To explore the protective effect of Klotho protein regulating the expression of cysteine protease-8 (Caspase-8)/Caspase-3 on neuronal apoptosis in rats with hypoxic-ischemic brain injury (HIBD). Methods A total of 60 HIBD rat models were constructed and divided into the model group and the low-dose, medium-dose, and high-dose Klotho groups, and another 15 neonatal rats were selected as the sham operation group. The low-dose, medium-dose and high-dose Klotho groups were treated with continuous ventricular injection of 10, 20 and 30mg/kg of Klotho protein for 5 days respectively, while the sham operation group and the model group were treated with the same volume of normal saline. The neurological function, apoptosis rate of nerve cells, Caspase-8, Caspase-3 and oxidative stress indicators in brain tissue of each group were compared after ventricular injection treatment. 〖WTHZ〗Results Compared with the sham operation group, the Longa method score and the apoptosis rate of nerve cells in the model group were significantly increased, and the levels of Caspase-8, Caspase-3, reactive oxygen species (ROS), and malondialdehyde (MDA) in the brain tissue were significantly elevated, and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) decreased significantly (all P < 0.05). Compared with the model group, the Longa score, the apoptosis rate of nerve cells, and the levels of Caspase-8, Caspase-3, ROS and MDA in brain tissue all decreased significantly the Klotho protein groups, and the activities of SOD and GSH-Px increased (all P< 0.05). Compared with the low-dose group of Klotho protein, the Longa method score, the apoptosis rate of nerve cells, and the levels of Caspase-8, Caspase-3, ROS, and MDA in brain tissue all decreased significantly in the medium and high-dose groups, and the activities of SOD and GSH-Px increased significantly (all P<0.05). Compared with the medium-dose group of Klotho protein, the Longa method score, the apoptosis rate of nerve cells, and the levels of Caspase-8, Caspase-3, ROS, and MDA in brain tissue all decreased significantly in the high-dose group, and the activities of SOD and GSH-Px increased significantly (all P<0.05). Conclusion The Klotho protein at a concentration of 30mg/kg can effectively inhibit the apoptosis of nerve cells in HIBD rats and improve the extension function of rats. The mechanism may be related to the inhibition of Caspase-8/Caspase-3 expression in rat brain tissue and the reduction of oxidative stress injury in brain tissue