Ischemic stroke is a major cause of neurological disability. Head-down positioning (HDP) has been proposed as a simple non-pharmacological intervention to improve cerebral perfusion after ischemic stroke. In this study, label-free quantitative proteomics was performed to characterize protein alterations associated with ischemic injury and HDP-mediated neuroprotection. Ischemic penumbral tissues were collected from male Sprague-Dawley rats subjected to middle cerebral artery occlusion (MCAO) followed by HDP intervention.