The axon initial segment (AIS) is involved in action potential initiation. Structural and biophysical characteristics of the AIS differ among cell types and/or brain regions, but the underlying mechanisms remain elusive. Using immunofluorescence and electrophysiological methods, combined with super-resolution imaging, we show in the developing nucleus magnocellularis of the chicken in both sexes that the AIS is refined in a tonotopic region-dependent manner. This process of AIS refinement differs among cells tuned to different frequencies. At hearing onset, the AIS was ;50 mm long with few voltage-gated sodium channels regardless of tonotopic region. However, after hatching, the AIS matured and displayed an ;20-mm-long structure with a significant enrichment of sodium channels responsible for an increase in sodium current and a decrease in spike threshold. Moreover, the shortening was more pronounced, while the accumulation of channels was not, in neurons tuned to higher frequency, creating tonotopic differences in the AIS. We conclude that AIS shortening is mediated by disassembly of the cytoskeleton at the distal end of the AIS, despite intact periodicity of the submembranous cytoskeleton across the AIS. Importantly, deprivation of afferent input diminished the shortening in neurons tuned to a higher frequency to a larger extent in posthatch animals, with little effect on the accumulation of sodium channels. Thus, cytoskeletal reorganization and sodium channel enrichment at the AIS are differentially regulated depending on tonotopic region, but work synergistically to optimize neuronal output in the auditory nucleus.