Voltage-gated Na(+) channel β-subunits are multifunctional molecules that modulate Na(+) channel activity and regulate cell adhesion, migration and neurite outgrowth. β-subunits including β4 are known to be highly concentrated in the nodes of Ranvier and axon initial segments in myelinated axons. Here we show diffuse β4 localization in striatal projection fibres using transgenic mice that express fluorescent protein in those fibres. These axons are unmyelinated, forming large, inhibitory fibre bundles. Furthermore, we report β4 dimer expression in the mouse brain, with high levels of β4 dimers in the striatal projection fascicles, suggesting a specific role of β4 in those fibres. Scn4b-deficient mice show a resurgent Na(+) current reduction, decreased repetitive firing frequency in medium spiny neurons and increased failure rates of inhibitory postsynaptic currents evoked with repetitive stimulation, indicating an in vivo channel regulatory role of β4 in the striatum.