Truncation mutations in the adenomatous polyposis coli (APC) gene are responsible for familial and sporadic colorectal cancer. APC is a multifunctional protein involved in cell migration, proliferation and differentiation. The APC protein forms specific clusters in the cell periphery that correlate with sites of active cell migration. Little is known about the molecular mechanisms that govern these clusters. Here, we identify a novel interaction of an N-terminal region of APC with the extreme C-terminal 300 amino acids of APC and also with itself. The latter interaction is phospho-sensitive and is enhanced by 14-3-3 (YWHA) protein. These interactions modulate the clustering of APC at the ends of membrane protrusions. Overexpressing this domain or inhibiting 14-3-3 proteins disperses APC clusters and leads to decreased cell migration. Moreover, deleting this domain from full-length APC results in less-dynamic clusters compared with wild-type APC. Our data indicate that this newly identified regions in the N-terminal third of APC contributes to the regulation of APC clusters, thus providing a molecular clue for how locally regulated phosphorylation events could mediate the dynamics of APC clusters and contribute to cell migration.