Gain-of-function of BK channels or knockout of their β4 subunit is associated with spontaneous epilepsy. Currently, efficacy of BK (α + β4) channel modulators in preventing epilepsy was never reported. Here, we show that martentoxin selectively inhibits BK (α + β4) channels by interaction with the extracellular loop of the BK β4 subunit (hβ4-loop) at a molar ratio 4:1 (hβ4-loop vs martentoxin). Residues Glu104, Glu122, Gln124, Lys125, and Glu128 of the hβ4-loop form hydrogen bonds with residues Asp5, Glu13, Lys20, Ser24, Gln26, Lys28, and Arg35 of martentoxin, by which martentoxin reduces the neuronal spiking frequency and increases interspike intervals. Intrahippocampal infusion of martentoxin significantly increases the latency time of seizure, reduces seizure duration and seizure numbers on pentylenetetrazole-induced presensitized rats, inhibits hippocampal hyperexcitability and c-Fos expression, and displays neuroprotective effects on hippocampal neurons. These results suggest that the BK (α + β4) channel is a novel therapeutic target of intractable epilepsy and martentoxin contributes to the rational drug design for epilepsy treatment.