Voltage-gated sodium channels (NaCh) are colocalized with isoforms of the membrane-skeletal protein ankyrin_G at axon initial segments, nodes of Ranvier, and postsynaptic folds of the mammalian neuromuscular junction. The role of ankyrin_G in directing NaCh localization to axon initial segments was evaluated by region-specific knockout of ankyrin_G in the mouse cerebellum. Mutant mice exhibited a progressive ataxia beginning around postnatal day P16 and subsequent loss of Purkinje neurons. In mutant mouse cerebella, NaCh were absent from axon initial segments of granule cell neurons, and Purkinje cells showed deficiencies in their ability to initiate action potentials and support rapid, repetitive firing. Neurofascin, a member of the L1CAM family of ankyrin-binding cell adhesion molecules, also exhibited impaired localization to initial segments of Purkinje cell neurons. These results demonstrate that ankyrin_G is essential for clustering NaCh and neurofascin at axon initial segments and is required for physiological levels of sodium channel activity.