Perinatal hypoxic‐ischemic (HI) brain injury is a major cause of permanent neurological dysfunction in children. An approach to study the treatment of neonatal HI encephalopathy that allows for neuroprotection is to investigate the states of tolerance to HI. Twenty‐four‐hour carotid‐artery ligation preconditioning established by delaying the onset of hypoxia for 24 hours after permanent unilateral carotid ligation rats markedly diminished the cerebral injury, however, the signaling mechanisms of this carotid‐artery ligation preconditioning in neonatal rats remain unknown. Ligation of the carotid artery 24 hours before hypoxia provided complete neuroprotection and produced improved performance on the Morris water maze compared with ligation performed 1 hour before hypoxia. Carotid artery ligation 6 hours before hypoxia produced intermediate benefit. The 24‐hour carotid‐artery ligation preconditioning was associated with a robust and sustained activation of a transcription factor, the cAMP response element–binding protein (CREB), on its phosphorylation site on Ser133. Intracerebroventricular infusions of antisense CREB oligodeoxynucleotides significantly reduced the 24‐hour carotid‐artery ligation–induced neuroprotection effects by decreasing CREB expressions. Pharmacological activation of the cAMP‐CREB signaling with rolipram 24 hours before hypoxia protected rat pups at behavioral and pathological levels by sustained increased CREB phosphorylation. This study suggests that 24‐hour carotid‐artery ligation preconditioning provides important mechanisms for potential pharmacological preconditioning against neonatal HI brain injury. Ann Neurol, 2004