Methamphetamine (MA), a highly abused amphetamine-like psychostimulant, has surged in popularity worldwide in the last decade. Repeated MA exposure has been shown to affect the alternative splice variant expression of large conductance Ca2+-activated K+ (BK) channels. It remains unclear whether MA affects BK channel activity. The present study investigated the effects of MA on BK channels in NG108-15 mouse neuroblastoma × rat glioma hybrid cells using whole-cell and cell-attached patch clamp techniques. In whole-cell recordings, the macroscopic K+ outward currents were inhibited by MA with an EC50 of 146 μM, but not affected by dopamine (DA). It implies that DA is not involved in the effects of MA on K+ outward currents. In cell-attached patches, MA significantly decreased BK channel activity. Moreover, MA significantly decreased the BK channel opener NS1619-evoked whole-cell K+ outward currents and BK channel activity. Finally, the effect of MA on membrane potential was examined by current-clamp configuration. MA caused membrane depolarization and application of NS1619 returned the depolarized potential to resting value. These findings suggest that MA might act as an inhibitor of BK channels, and thereby increase the neuronal excitability and enhance neurotransmitter release.