The axon initial segment is an excitable membrane highly enriched in voltage-gated sodium channels that integrates neuronal inputs and initiates action potentials. This study identifies Na_v1.6 as the voltage-gated sodium channel isoform at mature Purkinje neuron initial segments and reports an essential role for ankyrin-G in coordinating the physiological assembly of Na_v1.6, βIV spectrin, and the L1 cell adhesion molecules (L1 CAMs) neurofascin and NrCAM at initial segments of cerebellar Purkinje neurons. Ankyrin-G and βIV spectrin appear at axon initial segments by postnatal day 2, whereas L1 CAMs and Na_v1.6 are not fully assembled at continuous high density along axon initial segments until postnatal day 9. L1 CAMs and Na_v1.6 therefore do not initiate protein assembly at initial segments. βIV spectrin, Na_v1.6, and L1 CAMs are not clustered in adult Purkinje neuron initial segments of mice lacking cerebellar ankyrin-G. These results support the conclusion that ankyrin-G coordinates the physiological assembly of a protein complex containing transmembrane adhesion molecules, voltage-gated sodium channels, and the spectrin membrane skeleton at axon initial segments.