A modest autophagy benefits neuroprotection while an excessive autophagy leads to neuronal death after cerebral ischemia, but what governs an appropriate autophagy remains to be understood. Studies indicated that acetylation of histone H4 at lysine16 (H4K16ac) strongly modulated autophagic/lysosomal signaling pathway. Thus, this study was to investigate whether the autophagic neuronal injury could be alleviated by amending H4K16ac level after ischemic stroke. A rat model of middle cerebral artery occlusion (MCAO)/reperfusion was prepared to investigate dynamic variations between H4K16ac and autophagy at the penumbra. The results illustrated that the significantly elevated H4K16ac was coupled with dramatically promoted autophagic activity at 4 h after the insult, suggesting H4K16ac tightly controlled autophagic signaling. After that, H4K16ac level was altered by pretreatment with trichostatin A (TSA, a H4K16ac facilitator) and MG149 (a H4K16ac inhibitor), respectively. Four hours after MCAO/reperfusion, the penumbral tissues were obtained to detect the key proteins in autophagic/lysosomal pathway by western blot and immunofluorescence, respectively. Meanwhile, the infarct volume, neurological deficits, and neuron survival were assessed to evaluate the neurological outcomes. The results showed that TSA-promoted H4K16ac led to an excessively up-regulated autophagy resulting in autophagic/lysosomal dysfunction, as indicated by the accumulated autophagic substrates and exacerbated lysosomal inefficiency in neurons. By contrast, MG149-depressed H4K16ac significantly down-regulated autophagic activity and thereby restored the impaired autophagic flux. Consequently, the neurological injury was markedly alleviated in MCAO + MG149 group, compared with that in MCAO group. Our study suggests that the H4K16ac attenuation elicits neuroprotection against ischemic stroke by ameliorating autophagic/lysosomal dysfunction in neurons.