Sustained directionality is an essential component of successful chemotaxis. Here, the authors show that the G protein Gαi and the mammalian homologue of the Drosophila melanogaster polarity protein Inscuteable (known as MINSC) are important for the maintenance of polarity and directionality during neutrophil chemotaxis. They observed that Gαi (which is released from Gβγ following ligand binding) accumulates at the leading edge of the cell. Gαi interacts directly with AGS3 or LGN, which themselves are bound to MINSC, recruiting it to this part of the cell. Moreover, MINSC is bound to the polarity complex PAR3–PAR6–aPKC, and this interaction targets the complex to the leading edge, thus establishing polarity. Notably, MINSC-deficient neutrophils, or neutrophils in which aPKC was blocked, showed normal motility but lacked directionality in their chemotaxis.