Spreading depression (SD) is a pathological propagation of the neuronal and glial excitation underlying pathophysiology of the strokes and migraine. Previous studies indicated critical roles of glutamate in SD, since the glutamate receptor antagonist impairs SD propagation. Thus, it is hypothesized that sensitivity to the cortical SD is under the influence of glial glutamate transporter GLT-1 which is responsible for clearing >90% of extracellular glutamate. To address this, we generated conditional knockout mouse GFAP-Cre; GLT-1^flox/flox (GLT-1 cKO) showing significant reduction of GLT-1 protein without defects in its cytoarchitecture in the cerebral cortex. Field potential and cerebral blood flow measurement revealed that topical application of KCl to the visual cortex for 1 hour elicited more frequent and faster propagation of SD in the cortex of GLT-1 cKO than control, suggesting that GLT-1 determines the sensitivity to the cortical SD. To see the influence of propagating SD on the neural activity in the subcortical structures, we examined distribution of the cells expressing immediate early gene c-Fos 2.5 hours after onset of the cortical SD. Increase of c-Fos expression in control mice (GFAP-Cre; GLT-1^flox/+) was localized to the cortex and amygdala but not to the other subcortical structures as in the wild type mice. Despite the larger number of SD with faster velocity in the cortex, GLT-1 cKO did not show ectopic increase of c-Fos in the subcortical structures such as hippocampus and dorsal thalamus, suggesting the presence of functional barrier such as white matter which prevents SD from propagating outside the cortex.