The axon initial segment (AIS) is a unique axonal subdomain responsible for the generation of the neuronal action potential and the maintenance of the axon-dendritic functional polarity. Despite its importance, the mechanisms controlling AIS development and maintenance remain largely unknown. Here we show that the AIS microtubule cytoskeleton is composed of a pool of more stable, detergent resistant, microtubules. This AIS specific characteristic is conferred by the presence of CK2, an important regulator of microtubule stability, in the AIS during its development and maturation. We show that CK2alpha and CK2alpha' subunits concentrate at the AIS from its initial development, at the same time as pIkappaBalpha and ankyrinG. CK2 pharmacological inhibition or suppression of CK2alpha expression with nucleofected interference RNAs modifies microtubule characteristics throughout the neuron, changes KIF5C distribution, and impairs its own concentration at the AIS, as well as that of ankyrinG, ankyrinG-GFP, pIkappaBalpha and voltage gated sodium channels. Moreover, CK2alpha concentration at the AIS depends on IkappaBalpha phosphorylation by IKK and ankyrinG. In conclusion, our results demonstrate a mutual dependence of CK2, ankyrinG and pIkappaBalpha for their concentration at the axon initial segment, which is related to the specific characteristics of microtubules at the AIS.