AnkyrinG (ankG) is highly enriched in neurons at axon initial segments (AISs) where it clusters Na(+) and K(+) channels and maintains neuronal polarity. How ankG becomes concentrated at the AIS is unknown. Here, we show that as neurons break symmetry, they assemble a distal axonal submembranous cytoskeleton, comprised of ankyrinB (ankB), αII-spectrin, and βII-spectrin, that defines a boundary limiting ankG to the proximal axon. Experimentally moving this boundary altered the length of ankG staining in the proximal axon, whereas disruption of the boundary through silencing of ankB, αII-spectrin, or βII-spectrin expression blocked AIS assembly and permitted ankG to redistribute throughout the distal axon. In support of an essential role for the distal cytoskeleton in ankG clustering, we also found that αII and βII-spectrin-deficient mice had disrupted AIS. Thus, the distal axonal cytoskeleton functions as an intra-axonal boundary restricting ankG to the AIS.