Introduction: Although it is known that ischemic hyperglycolysis causes lactic acidosis and oxidative stress-induced ischemia/reperfusion injury, attempts to diminish these effects by targeting established mechanisms have not yet yielded improvement in clinical trials. Emerging evidence that gluconeogenesis (GNG) occurs in the brain suggests a novel manner in which ischemia, by shunting GNG intermediates, generates reactive oxygen species (ROS) and lactate, and thus also suggests a novel therapeutic target. We hypothesized that ischemia induces overactivation of the GNG enzymes phosphoenolpyruvake carboxykinase (PCK) 1&2, contributing to ROS and lactate-related damage. By inducing pharmacologic hypothermia (PH) with chlorpromazine and promethazine (C+P, FDA-approved), we tested the neuroprotective potential of suppressing these enzymes. Methods: Male Sprague-Dawley rats were subjected to 2 h intraluminal middle cerebral artery occlusion followed by 6 or 24 h reperfusion, upon which C+P (8mg/kg) and Troglitazone (TZD, a PCK inhibitor; 2.5mg/kg) were given intraperitoneally. Infarct volume and neurological deficit were determined. Additionally, primary rat and human neural cell lines were subjected to 2 h oxygen-glucose deprivation (OGD) and 6 or 24 h re-oxygenation, with or without C+P, TZD, or shRNA knockdown of PCK 1&2. Cell damage was assessed by colorimetric viability assay. In animal and cell culture models, ROS and lactate were measured. PCK 1&2 mRNA and protein levels were measured by real-time PCR and Western blotting. Levels of glucose and oxaloacetate (OAA), the GNG substrate of PCK, were obtained by biochemical assay. Cellular expressions of PCKs were determined by immunocytochemistry. Results: GNG occurred in the brain, and was dysregulated by ischemia: ROS and lactate levels correlated with increased neuronal PCK1&2 activity and with decreased OAA and glucose. C+P decreased oxidative/acidotic stress and overall brain injury by inhibiting PCK1&2 in neuronal and whole brain lysates. PCK1&2 were expressed primarily in neurons, but the effects of stroke on PCKs were largest in astrocytes. Conclusion: Our findings implicate GNG in ischemia/reperfusion pathogenesis and establish a new target of C+P neuroprotection.