Previously, we showed that a hierarchy of spectrin cytoskeletal proteins maintains nodal Na+ channels (Liu et al., 2020). Here, using mice lacking β1, β4, or β1/β4 spectrins, we show this hierarchy does not function at axon initial segments (AIS). Although β1 spectrin, together with AnkyrinR (AnkR), compensates for loss of nodal β4 spectrin, it cannot compensate at AIS. We show AnkR lacks the domain necessary for AIS localization. Whereas loss of β4 spectrin causes motor impairment and disrupts AIS, loss of β1 spectrin has no discernable effect on central nervous system structure or function. However, mice lacking both neuronal β1 and β4 spectrin show exacerbated nervous system dysfunction compared to mice lacking β1 or β4 spectrin alone, including profound disruption of AIS Na+ channel clustering, progressive loss of nodal Na+ channels, and seizures. These results further define the important role of AIS and nodal spectrins for nervous system function.