Background and Purpose-Erythropoietin (EPO) confers potent neuroprotection against ischemic injury. However, treatment for stroke requires high doses and multiple administrations of EPO, which may cause deleterious side effects due to its erythropoietic activity. This study identifies a novel nonerythropoietic mutant EPO and investigates its potential neuroprotective effects and underlying mechanism in an animal model of cerebral ischemia. Methods-We constructed a series of mutant EPOs, each containing a single amino acid mutation within the erythropoietic motif, and tested their erythropoietic activity. Using cortical neuronal cultures exposed to -N--methyl--d--aspartate neurotoxicity and a murine model of transient middle cerebral artery occlusion, neuroprotection and neurofunctional outcomes were assessed as well as activation of intracellular signaling pathways. Results-The serine to isoleucine mutation at position 104 (S104I-EPO) completely abolished the erythropoietic and -platelet--stimulating activity of EPO. Administration of S104-I--EPO significantly inhibited -N--methyl-d--aspartate--induced neuronal death in primary cultures and protected against cerebral infarction and neurological deficits with an efficacy similar to that of -wild--type EPO. Both S104-I--EPO and -wild--type EPO activated similar prosurvival signaling pathways such as phosphatidylinositol 3-kinase/AKT, -mitogen--activated protein kinase/extracellular -signal--regulated kinase 1/2, and STAT5. Inhibition of phosphatidylinositol 3-kinase/AKT or -mitogen--activated protein kinase/extracellular -signal-regulated kinase 1/2 signaling pathways significantly attenuated the neuroprotective effects of S104-I--EPO, indicating that activation of these pathways underlies the neuroprotective mechanism of mutant EPO against cerebral ischemia. Conclusions-S104-I--EPO confers neuroprotective effects comparable to those of -wild--type EPO against ischemic brain in-jury with the added benefit of lacking erythropoietic and -platelet--stimulating side effects. Our novel findings suggest that the nonerythropoietic mutant EPO is a legitimate candidate for ischemic stroke intervention. (Stroke. 2012; 43: 3071-3077.)