Epilepsy is the results from the imbalance between inhibition and excitation in neural circuits, which is mainly treated by some chemical drugs with side effects. Gain-of-function of BK channels or knockout of its β4 subunit associates with spontaneous epilepsy. Currently, few reports were published about the efficacy of BK(α + β4) channel modulators in epilepsy prevention. Charybdotoxin is a non-specific inhibitor of BK and other K+ channels. Here, by nuclear magnetic resonance (NMR) and other biochemical techniques, we found that charybdotoxin might interact with the extracellular loop of human β4 subunit (i.e., hβ4-loop) of BK(α + β4) channel at a molar ratio 4:1 (hβ4-loop vs. charybdotoxin). Charybdotoxin enhanced its ability to prevent K+ current of BK(α + β4 H101Y) channel. The charybdotoxin Q18F variant selectively reduced the neuronal spiking frequency and increased interspike intervals of BK(α + β4) channel by π-π stacking interactions between its residue Phe18 and residue His101 of hβ4-loop. Moreover, intrahippocampal infusion of charybdotoxin Q18F variant significantly increased latency time of seizure, reduced seizure duration and seizure numbers on pentylenetetrazole-induced pre-sensitized rats, inhibited hippocampal hyperexcitability and c-Fos expression, and displayed neuroprotective effects on hippocampal neurons. These results implied that charybdotoxin Q18F variant could be potentially used for intractable epilepsy treatment by therapeutically targeting BK(α + β4) channel.