Astrocytes regulate neural excitability via the spatial potassium (K⁺) buffering mediated by inwardly rectifying Kir4.1 channels. In this study, we examined the effects of fluorocitrate (FC), a selective inhibitor of the astrocyte TCA cycle, on the expression of astrocytic Kir4.1 channels and the seizure susceptibility to pentylenetetrazole (PTZ) in rats. Microinjection of FC (1 nmol) into the right lateral ventricle caused no spontaneous seizures, but the number of the glial fibrillary acidic protein (GFAP)-positive astrocytes was decreased in the perirhinal-ectorhinal cortex, piriform cortex (PirC), basolateral amygdala (BLP), CA2 area and dentate gyrus (DG) of hippocampus. In addition, FC reduced Kir4.1 expression in PirC, CA2, and DG. When the susceptibility of rats to PTZ (40 mg/kg, i.p.)-induced seizures was evaluated, a significant increase in seizure intensity, an increased seizure incidence, and a tendency of delay in seizure onset were observed following the pretreatment of rats with FC (1 nmol, i.c.v.). Furthermore, PTZ-induced expression of Fos protein, a biological marker of neural excitation, was significantly increased in BLP, basomedial amygdala, and DG by the pretreatment with FC. These results suggest that the dysfunction of astrocytic Kir4.1 channels by FC elevates the susceptibility to PTZ seizures via hyperactivating the amygdala and hippocampal neurons.