We previously reported that uridine blocked glucose deprivation-induced death of immunostimulated astrocytes by preserving ATP levels. Uridine phosphorylase (UPase), an enzyme catalyzing the reversible phosphorylation of uridine, was involved in this effect. Here, we tried to expand our previous findings by investigating the uridine effect on the brain and neurons using in vivo and in vitro ischemic injury models. Orally administrated uridine (50-200 mg/kg) reduced middle cerebral artery occlusion (1.5 h)/reperfusion (22 h)-induced infarct in mouse brain. Additionally, in the rat brain subjected to the same ischemic condition, UPase mRNA and protein levels were up-regulated. Next, we employed glucose deprivation-induced hypoglycemia in mixed cortical cultures of neurons and astrocytes as an in vitro model. Cells were deprived of glucose and, two hours later, supplemented with 20 mM glucose. Under this condition, a significant ATP loss followed by death was observed in neurons but not in astrocytes, which were blocked by treatment with uridine in a concentration-dependent manner. Inhibition of cellular uptake of uridine by S-(4-nitrobenzyl)-6-thioinosine blocked the uridine effect. Similar to our in vivo data, UPase expression was up-regulated by glucose deprivation in mRNA as well as protein levels. Additionally, 5-(phenylthio)acyclouridine, a specific inhibitor of UPase, prevented the uridine effect. Finally, the uridine effect was shown only in the presence of astrocytes. Taken together, the present study provides the first evidence that uridine protects neurons against ischemic insult-induced neuronal death, possibly through the action of UPase. © 2008 Mary Ann Liebert, Inc.