The axon initial segment (AIS) is a compartment that serves as a molecular barrier to achieve axon-dendrite differentiation. Distribution of specific proteins during early neuronal development has been proposed to be critical for AIS construction. However, it remains unknown how these proteins are specifically targeted to the proximal axon within this limited time period. Here, we reveal spatiotemporal regulation driven by the microtubule (MT)-based motor KIF3A/B/KAP3 that transports TRIM46, influenced by a specific MARK2 phosphorylation cascade. In the proximal part of the future axon under low MARK2 activity, the KIF3/KAP3 motor recognizes TRIM46 as cargo and transports it to the future AIS. In contrast, in the somatodendritic area under high MARK2 activity, KAP3 phosphorylated at serine 60 by MARK2 cannot bind with TRIM46 and be transported. This spatiotemporal regulation between KIF3/KAP3 and TRIM46 under specific MARK2 activity underlies the specific transport needed for axonal differentiation.