Seizure is a serious complication of stroke, indicating poor prognosis. Notch signaling is associated with neuronal activity. Inhibition of Notch signaling suppresses seizure activity induced by kainic acid. The present study investigated the effect of the Notch inhibitor, DAPT, alone or in combination with mild hypothermia, on post-stroke seizures. A global cerebral ischemia (GCI) model was performed in Sprague Dawley (SD) male rats. Seizure activity was evaluated by the frequency of seizure attacks, seizure severity scores, and seizure discharges. Without any intervention, seizures occurred intensively between 24h and 48h following GCI. Seizure activity was confirmed using EEG monitoring. The expression of Notch intracellular domains (NICD) 1 and 2 were up-regulated in the cerebral cortex and hippocampus following GCI. DAPT was injected into the hippocampus of the rats to inhibit local Notch signaling. Active whole-body cooling was performed to maintain the core temperatures of rats at 33.5°C (mild hypothermia). Mild hypothermia and DAPT synergistically inhibited NICD 1 and 2 up-regulation, and post-stroke seizures. GCI augmented excitatory synaptic neurotransmission by up-regulating glutamate receptor subunits (GluN2A, GluA1) and the cotransporter, NKCC1, but attenuated inhibitory synaptic neurotransmission by down-regulating gamma amino acid, butyric acid (GABA), and the cotransporter, KCC2. DAPT treatment normalized the homeostasis of excitatory and inhibitory synaptic neurotransmission, suggesting that aberrant activation of Notch signaling is involved in post-stroke seizures. The present study adds to the further understanding of the pathogenesis of post-stroke seizures and the improvement of the treatment provided with hypothermia.