Prothymosin alpha (ProTα), a nuclear protein devoid of signal sequence, has been shown to possess a number of cellular functions including cell survival. Most recently, we demonstrated that ProTα is localized in the nuclei of neurons, while it is found in both nuclei and cytoplasm in the astrocytes and microglia of adult brain. However, the cell type-specific non-classical release of ProTα under cerebral ischemia is yet unknown. In this study, we report that ProTα is non-classically released along with S100A13 from neurons in the hippocampus, striatum and somatosensory cortex at 3 h after cerebral ischemia, but amlexanox (an anti-allergic compound) reversibly blocks this neuronal ProTα release. We found that none of ProTα is released from astrocytes and microglia under ischemic stress. Indeed, ProTα intensity is increased gradually in astrocytes and microglia through 24 h after the cerebral ischemia. Interestingly, Z-Val-Ala-Asp fluoromethyl ketone, a caspase 3 inhibitor, pre-treatment induces ProTα release from astrocytes in the ischemic brain, but this release is reversibly blocked by amlexanox. However, Z-Val-Ala-Asp fluoromethyl ketone as well as amlexanox has no effect on ProTα distribution in microglia upon cerebral ischemia. Taken together, these results suggest that only neurons have machineries to release ProTα upon cerebral ischemic stress in vivo.