Non-muscle myosin IIA (NMII-A) and the tumor suppressor Lgl1 play a central role in the polarization of migrating cells. Mammalian Lgl1 interacts directly with NMII-A, inhibiting its ability to assemble into filaments in vitro. Lgl1 also regulates the cellular localization of NMII-A, the maturation of focal adhesions and cell migration. In Drosophila, phosphorylation of Lgl affects its association with the cytoskeleton. Here we show that phosphorylation of mammalian Lgl1 by aPKCζ prevents its interaction with NMII-A both in vitro and in vivo, and affects its inhibition on NMII-A filament assembly. Phosphorylation of Lgl1 affects its cellular localization and is important for the cellular organization of the acto-NMII cytoskeleton. We further show that Lgl1 forms two distinct complexes in vivo, Lgl1-NMIIA and Lgl1-Par6α-aPKCζ and that the complexes formation is affected by the phosphorylation state of Lgl1. The complex Lgl1-Par6α-aPKCζ resides in the leading edge of the cell. Finally, we show that aPKCζ and NMII-A compete to bind directly to Lgl1 via the same domain. These results provide new insights into the mechanism regulating the interaction between Lgl1, NMII-A, Par6α, and aPKCζ in polarized migrating cells.