AbstractVoltage‐gated potassium (Kv) channels are critical to regulation of neurotransmitter release throughout the nervous system but the roles and identity of the subtypes involved remain unclear. Here we show that Kv3 channels regulate transmitter release at the mouse neuromuscular junction (NMJ). Light‐ and electron‐microscopic immunohistochemistry revealed Kv3.3 and Kv3.4 subunits within all motor nerve terminals of muscles examined [transversus abdominus, lumbrical and flexor digitorum brevis (FDB)]. To determine the roles of these Kv3 subunits, intracellular recordings were made of end‐plate potentials (EPPs) in FDB muscle fibres evoked by electrical stimulation of tibial nerve. Tetraethylammonium (TEA) applied at low concentrations (0.05–0.5 mm), which blocks only a few known potassium channels including Kv3 channels, did not affect muscle fibre resting potential but significantly increased the amplitude of all EPPs tested. Significantly, this effect of TEA was still observed in the presence of the large‐conductance calcium‐activated potassium channel blockers iberiotoxin (25–150 nm) and Penitrem A (100 nm), suggesting a selective action on Kv3 subunits. Consistent with this, 15‐µm 4‐aminopyridine, which blocks Kv3 but not large‐conductance calcium‐activated potassium channels, enhanced evoked EPP amplitude. Unexpectedly, blood‐depressing substance‐I, a toxin selective for Kv3.4 subunits, had no effect at 0.05–1 µm. The combined presynaptic localization of Kv3 subunits and pharmacological enhancement of EPP amplitude indicate that Kv3 channels regulate neurotransmitter release from presynaptic terminals at the NMJ.