Prothymosin-alpha (ProTalpha) causes a switch in cell death mode from necrosis to neurotrophin-reversible apoptosis in primary cultured cortical neurons. In the present study, post-ischemic administration (3 or 24 h, intravenously) of recombinant mouse ProTalpha without neurotrophins completely prevented ischemia-induced retinal damage accompanying necrosis and apoptosis, as well as dysfunction assessed by electroretinogram. Treatments with anti-erythropoietin (EPO) or brain-derived neurotrophic factor (BDNF) immunoglobulin G (IgG) reversed ProTalpha-induced inhibition of apoptosis. ProTalpha upregulated retinal EPO and BDNF levels in the presence of ischemia. Moreover, intravitreous administration of anti-ProTalpha IgG or an antisense oligodeoxynucleotide for ProTalpha accelerated ischemia-induced retinal damage. We also observed that ischemia treatment caused a depletion of ProTalpha from retinal cells. Altogether, these results suggest that the systemic administration of ProTalpha switches ischemia-induced necrosis to apoptosis, which in turn is inhibited by neurotrophic factors upregulated by ProTalpha and ischemia. ProTalpha released upon ischemic stress was found to have a defensive role in retinal ischemia.