Hydroxysafflor yellow A (HSYA) has been approved clinically for treating cardiac patients in China since 2005. Recent studies have indicated that HSYA may be neuroprotective at 24 h in experimental stroke models. Autophagy is a vital degradation pathway of damaged intracellular macromolecules or organelles to maintain homeostasis in physiological or pathological conditions. The purpose of this study is to investigate the neuroprotection of HSYA at 72 h and its mechanism via activating the autophagy pathway using an acute ischemic-reperfusion stroke rat model. Rats were treated with HSYA (2 mg/kg) during 90 min middle cerebral artery occlusion/72 h reperfusion by intravenous administration at four different time points (15 min post-ischemia, 15 min, 24 h, and 48 h post reperfusion), mimicking the potential treatment for acute ischemic stroke. HSYA administration reduced infarction volume and improved various neurological functions at 72 h of reperfusion. The possible molecular mechanism was investigated. We found that HSYA activated the AKT-autophagy pathway in penumbra tissue, which occurred in neuronal-specific cells. Moreover, blocking the AKT-autophagy pathway by an AKT inhibitor abolished HSYA-induced neuroprotection after cerebral ischemia. HSYA may be a promising drug for treating acute ischemic stroke and the AKT-dependent autophagy pathway contributes to the HSYA-afforded neuroprotection.