The phosphoinositide phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] regulates the activity of many actin-binding proteins and as such is an important modulator of cytoskeleton organization during cell migration, for example. In migrating cells actin remodeling is tightly regulated and localized; therefore, how the PI(4,5)P2 level is spatially and temporally regulated is crucial to understanding how it controls cell migration. Here we show that the LIM protein Ajuba contributes to the cellular regulation of PI(4,5)P2 levels by interacting with and activating the enzymatic activity of the PI(4)P 5-kinase (PIPKIα), the predominant enzyme in the synthesis of PI(4,5)P2, in a migration stimulus-regulated manner. In migrating primary mouse embryonic fibroblasts (MEFs) from Ajuba−/− mice the level of PI(4,5)P2 was decreased with a corresponding increase in the level of the substrate PI(4)P. Reintroduction of Ajuba into these cells normalized PI(4,5)P2 levels. Localization of PI(4,5)P2 synthesis and PIPKIα in the leading lamellipodia and membrane ruffles, respectively, of migrating Ajuba−/− MEFs was impaired. In vitro, Ajuba dramatically activated the enzymatic activity of PIPKIα while inhibiting the activity of PIPKIIβ. Thus, in addition to its effects upon Rac activity Ajuba can also influence cell migration through regulation of PI(4,5)P2 synthesis through direct activation of PIPKIα enzyme activity.