Pharmacologic approaches to activate K+ channels represent an emerging strategy to regulate membrane excitability. Here we report the identification and characterization of a lipid soluble toxin, mallotoxin (rottlerin), which potently activates the large conductance voltage and Ca2+-activated K+ channel (BK) expressed in a heterologous expression system and human vascular smooth muscle cells, shifting the conductance/voltage relationship by >100 mV. Probing the mechanism of action, we discover that the BK channel can be activated in the absence of divalent cations (Ca2+, Mg2+), suggesting that the mallotoxin mechanism of action involves the voltage-dependent gating of the channel. Mallotoxin-activated channels remain incrementally sensitive to Ca2+ and beta subunits. In comparison to other small hydrophobic poisons, anesthetic agents, and protein toxins that inhibit ion channel activity, mallotoxin potently activates channel activity. In certain respects, mallotoxin acts as a BK channel beta1 subunit mimetic, preserving BK channel Ca2+ sensitivity yet adjusting the set-point for BK channel activation to a more hyperpolarized membrane potential.