All class I phosphoinositide 3-kinases (PI3Ks) associate tightly with regulatory subunits through interactions that have been thought to be constitutive. PI3Kγ is key to the regulation of immune cell responses activated by G protein-coupled receptors (GPCRs). Remarkably we find that PKCβ phosphorylates Ser582 in the helical domain of the PI3Kγ catalytic subunit p110γ in response to clustering of the high-affinity IgE receptor (FcεRI) and/or store-operated Ca²⁺- influx in mast cells. Phosphorylation of p110γ correlates with the release of the p84 PI3Kγ adapter subunit from the p84-p110γ complex. Ser582 phospho-mimicking mutants show increased p110γ activity and a reduced binding to the p84 adapter subunit. As functional p84-p110γ is key to GPCR-mediated p110γ signaling, this suggests that PKCβ-mediated p110γ phosphorylation disconnects PI3Kγ from its canonical inputs from trimeric G proteins, and enables p110γ to operate downstream of Ca²⁺ and PKCβ. Hydrogen deuterium exchange mass spectrometry shows that the p84 adaptor subunit interacts with the p110γ helical domain, and reveals an unexpected mechanism of PI3Kγ regulation. Our data show that the interaction of p110γ with its adapter subunit is vulnerable to phosphorylation, and outline a novel level of PI3K control.