G protein-gated inwardly rectifying potassium (GIRK) channels are important inhibitory regulators of neuronal excitability in central nervous system, and the impairment of GIRK channel function has been reported to be associated with the susceptibility of epilepsy. However, the dynamics of GIRK channels in the pathogenesis of epilepsy are still unclear. In this study, our results showed that cyclothiazide, a potent convulsant, dose dependently increased the epileptiform bursting activities and suppressed the baclofen induced GIRK currents. In addition, TPQ, a selective GIRK antagonist, significantly decreased the total inwardly rectifying potassium (Kir) current, and increased the neuronal epileptiform activities. In contrast, ML297, a potent and selective GIRK channel agonist, reversed the cyclothiazide induced decrease of GIRK currents and the increase of neuronal excitability in cultured hippocampal neurons. Further investigation revealed that GIRK1, but not GIRK2, played a key role in suppressing epileptic activities. Finally, in pilocarpine mice seizure model, we demonstrated that ML297 significantly suppressed the seizure behavior. In summary, our current results indicate that GIRK channels, especially GIRK1-containing channels, are involved in epileptic activities and ML297 has a potential antiepileptic effect.