AbstractIsoleucine is a branched chain amino acid. The role of isoleucine in cerebral ischemia–reperfusion injury remains unclear. Here, we show that the concentration of isoleucine is decreased in cerebrospinal fluid in a rat model of cerebral ischemia–reperfusion injury, the rat middle cerebral artery occlusion (MCAO). To our surprise, the level of intraneuronal isoleucine is increased in an in vitro model of cerebral ischemia injury, the oxygen–glucose deprivation (OGD). We found that the increased activity of LAT1, an L‐type amino acid transporter 1, leads to the elevation of intraneuronal isoleucine after OGD insult. Reducing the level of intraneuronal isoleucine promotes cell survival after cerebral ischemia–reperfusion injury, but supplementing isoleucine aggravates the neuronal damage. To understand how isoleucine promotes ischemia‐induced neuronal death, we reveal that isoleucine acts upstream to reduce the expression of CBFB (core binding factor β, a transcript factor involved in cell development and growth) and that the phosphatase PTEN acts downstream of CBFB to mediate isoleucine‐induced neuronal damage after OGD insult. Interestingly, we demonstrate that direct‐current stimulation reduces the level of intraneuronal isoleucine in cortical cultures subjected to OGD and that transcranial direct‐current stimulation (tDCS) decreases the cerebral infarct volume of MCAO rat through reducing LAT1‐depencent increase of intraneuronal isoleucine. Together, these results lead us to conclude that LAT1 over activation‐dependent isoleucine‐CBFB‐PTEN signal transduction pathway may mediate ischemic neuronal injury and that tDCS exerts its neuroprotective effect by suppressing LAT1 over activation‐dependent signalling after cerebral ischemia–reperfusion injury.