Differential neurological outcomes due to prostaglandin E2 activating G-protein-coupled prostaglandin E (EP) receptors have been observed. Here, we investigated the action of the EP4/EP3 agonist 1-hydroxyPGE1 (1-OHPGE1) in modulating transient ischemic brain damage. C57BL/6 mice were pretreated 50 min before transient occlusion of the middle cerebral artery with an intraventricular injection of 1-OHPGE1 (0.1, 0.2, 2.0 nmol/0.2 µL). Brain damage 4 days after reperfusion, as estimated by infarct volume, was significantly reduced by more than 19% with 1-OHPGE1 in the two higher-dose groups (P < 0.05). To further address whether protection also was extended to neurons, primary mouse cultured neuronal cells were exposed to N-methyl-d-aspartate. Co-treatment with 1-OHPGE1 resulted in significant neuroprotection (P < 0.05). To better understand potential mechanisms of action and to test whether changes in cyclic adenosine monophosphate (cAMP) levels and downstream signaling would be neuroprotective, we measured cAMP levels in primary neuronal cells. Brief exposure to 1-OHPGE1 increased cAMP levels more than twofold and increased the phosphorylation of extracellular-regulated kinases at positions Thr-202/Tyr-204. In a separate cohort of animals, 1-OHPGE1 at all doses tested produced no significant effect on the physiological parameters of core body temperature, mean arterial pressure and relative cerebral blood flow observed following drug treatment. Together, these results suggest that modulation of PGE2 receptors that increase cAMP levels and activate extracellular-regulated kinases 1/2 caused by treatment with 1-OHPGE1 can be protective against neuronal injury induced by focal ischemia.