Intranasal (IN) administration appears to be a suitable route for clinical use as it allows direct delivery of bioactive molecules to the central nervous system, reducing systemic exposure and sides effects. Nevertheless, only some molecules can be transported to the brain from the nasal cavity. This led us to compare the efficiency of an IN, intravenous (IV), and intraperitoneal (IP) administration of pituitary adenylate cyclase-activating polypeptide (PACAP) after transient or permanent middle cerebral artery occlusion (MCAO) in C57BL/6 mice. The results show that the neuroprotective effect of PACAP is much more efficient after IN administration than IV injection while IP injection had no effect. IN administration of PACAP reduced the infarct volume when injected within 6 h after the reperfusion and improved functional recovery up to at least 1 week after the ischemia.