Phosphoinositide 3-kinase (PI3K) is regulated by receptor tyrosine kinases (RTKs), G protein– coupled receptors (GPCRs), and small GTPases such as Rac1 and Rab5. Our lab previously identified two residues (Gln596 and Ile597) in the helical domain of the catalytic subunit (p110) of PI3K whose mutation disrupts binding to Rab5. To better define the Rab5–p110 interface, we performed alanine-scanning mutagenesis and analyzed Rab5 binding with an in vitro pulldown assay with GST-Rab5GTP. Of the 35 p110 helical domain mutants assayed, 11 disrupted binding to Rab5 without affecting Rac1 binding, basal lipid kinase activity, or G-stimulated kinase activity. These mutants defined the Rab5-binding interface within p110 as consisting of two perpendicular -helices in the helical domain that are adjacent to the initially identified Gln596 and Ile597 residues. Analysis of the Rab5–PI3K interaction by hydrogen-deuterium exchange MS identified p110 peptides that overlap with these helices; no interactions were detected between Rab5 and other regions of p110 or p85. Similarly, the binding of Rab5 to isolated p85 could not be detected, and mutations in the Ras-binding domain (RBD) of p110 had no effect on Rab5 binding. Whereas soluble Rab5 did not affect PI3K activity in vitro, the interaction of these two proteins was critical for chemotaxis, invasion, and gelatin degradation by breast cancer cells. Our results define a single, discrete Rab5-binding site in the p110 helical domain, which may be useful for generating inhibitors to better define the physiological role of Rab5–PI3K coupling in vivo.