In many neuron types, the axon initial segment (AIS) has the lowest threshold for action potential generation. Its active properties are determined by the targeted expression of specific voltage-gated channel subunits. We show that the Na⁺ channel Na_V1.6 displays a striking aggregation at the AIS of cortical neurons. To assess the functional role of this subunit, we used Scn8a^med mice that are deficient for Na_V1.6 subunits but still display prominent Na⁺ channel aggregation at the AIS. In CA1 pyramidal cells from Scn8a^med mice, we found a depolarizing shift in the voltage dependence of activation of the transient Na⁺ current (I_NaT), indicating that Na_V1.6 subunits activate at more negative voltages than other Na_V subunits. Additionally, persistent and resurgent Na⁺ currents were significantly reduced. Current-clamp recordings revealed a significant elevation of spike threshold in Scn8a^med mice as well as a shortening of the estimated delay between spike initiation at the AIS and its arrival at the soma. In combination with simulations using a realistic computer model of a CA1 pyramidal cell, our results imply that a hyperpolarized voltage dependence of activation of AIS Na_V1.6 channels is important both in determining spike threshold and localizing spike initiation to the AIS. In addition to altered spike initiation, Scn8a^med mice also showed a strongly reduced spike gain as expected with combined changes in persistent and resurgent currents and spike threshold. These results suggest that Na_V1.6 subunits at the AIS contribute significantly to its role as spike trigger zone and shape repetitive discharge properties of CA1 neurons.