Mounting attention has been focused on defects in macroautophagy/autophagy and the autophagy-lysosomal pathway (ALP) in cerebral ischemia. TFEB (transcription factor EB)-mediated induction of ALP has been recently considered as the common mechanism in ameliorating the pathological lesion of myocardial ischemia and neurodegenerative diseases. Here we explored the vital role of TFEB in permanent middle cerebral artery occlusion (pMCAO)-mediated dysfunction of ALP and ischemic insult in rats. The results showed that ALP function was first enhanced in the early stage of the ischemic process, especially in neurons of the cortex, and this was accompanied by increased TFEB expression and translocation to the nucleus, which was mediated at least in part through activation by PPP3/calcineurin. At the later stages of ischemia, a gradual decrease in the level of nuclear TFEB was coupled with a progressive decline in lysosomal activity, accumulation of autophagosomes and autophagy substrates, and exacerbation of the ischemic injury. Notably, neuron-specific overexpression of TFEB significantly enhanced ALP function and rescued the ischemic damage, starting as early as 6 h and even lasting to 48 h after ischemia. Furthermore, neuron-specific knockdown of TFEB markedly reversed the activation of ALP and further aggravated the neurological deficits and ischemic outcome at the early stage of pMCAO. These results highlight neuronal-targeted TFEB as one of the key players in the pMCAO-mediated dysfunction of ALP and ischemic injury, and identify TFEB as a promising target for therapies aimed at neuroprotection in cerebral ischemia. Abbreviations: AAV, adeno-associated virus; AIF1/IBA1, allograft inflammatory factor 1; ALP, autophagy-lysosomal pathway; CQ, chloroquine; CTSB, cathepsin B; CTSD, cathepsin D; CsA, cyclosporin A; GFAP, glial fibrillary acidic protein; LAMP, lysosomal-associated membrane protein; LC3, microtubule-associated protein 1 light chain 3; MAP2, microtubule-associated protein 2; mNSS, modified Neurological Severity Score; MTOR, mechanistic target of rapamycin kinase; OGD, oxygen and glucose deprivation; pMCAO, permanent middle cerebral artery occlusion; RBFOX3/NeuN, RNA binding fox-1 homolog 3; SQSTM1, sequestosome1; TFEB, transcription factor EB; TTC, 2,3,5-triphenyltetrazolium chloride.