Objective Formononetin (FOR), a traditional Chinese medicine, has been widely used for nerve protection and nerve function rehabilitation after cerebral stroke. However, the role of FOR in autophagic lysosome function in cerebral ischemia-reperfusion damage has not been investigated. This study aimed to explore whether the therapeutic benefits of FOR were influenced by the regulation of autophagy flux.Methods Male Sprague-Dawley rats were separated into sham, model, and MCAO+FOR (30 mg/kg) groups after undergoing middle cerebral artery occlusion (MCAO) and ischemia-reperfusion (I/R). Then, the brain tissues in the ischemic penumbra were obtained to detect the proteins in autophagic/lysosomal pathway with antibodies of Beclin-1, LC3, SQSTM1/P62, Ubiquitin, LAMP-2, Cathepsin B (CTSB) and Cathepsin D (CTSD) by Western blot and immunofluorescence, respectively. Meanwhile, the therapeutic effectiveness was evaluated by measuring infarct volume, neurological impairments, and neuronal necrosis.Results The findings of this study demonstrate that FOR treatment exhibits a dual effect by enhancing the autophagic activities of Beclin-1 and LC3 in neurons, while simultaneously improving the autophagic clearance function, as evidenced by reinforced lysosomal activities of LAMP-2, CTSB, and CTSD, as well as reduced autophagic accumulation of Ubiquitin and P62 in the MCAO+FOR group compared to the MCAO group. Additionally, 7 d of FOR treatment dramatically reduced neurological deficits, infarct volume, and neuronal death caused by cerebral ischemia.Conclusion These findings suggest that the neuroprotective mechanism of FOR therapy in accelerating recovery from ischemic stroke may involve the increase of autophagy flux in the penumbra.