The axonal initial segment (AIS) is a crucial neuronal subdomain that coordinates the axo-dendritic polarity establishment and maintenance and constitutes the site for action potential (AP) initiation. Its integrity relies on proteins like AnkyrinG (AnkG) which, by organizing the cytoskeleton, anchor the ionic channels necessary for AP generation. Many AIS proteins are linked to neurodevelopmental disorders (NDD) like epilepsy or autism and the AIS proteome deciphering is warranted to provide insight into novel therapeutic targets for the treatment of human diseases of the nervous system.Planar cell polarity (PCP) signaling controls tissue morphogenesis and cell polarity through cytoskeleton reorganization, and has important functions during nervous system development. Mutations in PCP genes lead to NDD, such as neural tube closure defects or epilepsy, but their molecular role and function in neurons are still obscure.In addition to Prickle2, we discovered another core PCP protein that accumulates at the AIS in vitro during neuronal development but also in vivo. We observe its colocalization with specific AIS members, such as TRIM46, implying a potential impact in the stabilization of microtubules (MT). The downregulation of this novel AIS component impaired AIS formation by altering the distribution and levels of AnkG and other AIS markers. We could also observe a disorganization of AIS MT bundles that is eventually associated with a strong reduction of the AIS component of the AP.Our data identify a new PCP player in AIS formation and reveal novel mechanisms in axonal initial segment formation, and function.