Background and Purpose: Isoleucine is a branched-chain amino acid serving as an essential nutrient resource and metabolic. However， its role in cerebral ischemic stroke remains unknown. Experimental Approach: Middle cerebral artery occlusion (MCAO) was used to mimic in vivo model of stroke. Oxygen-glucose deprivation insult (OGD) was used to injure cultured cortical neurons. High-Performance Liquid Chromatography (HPLC) was used to measure the level of isoleucine. A western blot assay and immunofluorescent staining were used to measure the level of CBFB and PTEN. TTC staining was used to measure the infarct size. Cell death and viability were assessed by LDH and CCK8 assays. DCS was used to stimulate cortical neurons. tDCS was used to stimulate the cortex. Key Results: Extraneuronal isoleucine is decreased and intraneuronal isoleucine is increased after rat cerebral I/R injury. Reducing intraneuronal isoleucine via inhibition of its transporter, LAT1 promotes neuronal survival whereas supplementing isoleucine aggravates neuronal damage. Isoleucine downregulates the expression of CBFB, and that acts upstream of PTEN to mediate isoleucine-induced neuronal damage after OGD insult. To identify the therapeutic approach that suppresses the ischemia-induced increase of intraneuronal isoleucine, we tested the effect of tDCS on isoleucine. Our data suggest that Cathodal tDCS can reduce cerebral infarct size. And such neuroprotection is mediated through reducing LAT1-dependent increase of intraneuronal isoleucine. Conclusions and Implications: This study identifies LAT1- dependent increase of intraneuronal isoleucine promotes neuronal death after rat cerebral I/R injury. Our results indicate that tDCS protects against rat cerebral I/R injury through regulating LAT1-isoleucine-CBFB-PTEN signaling.