Rho GTPases regulate actin dynamics and cell motility, and their hyperactivation drives cancer metastasis1. P-Rex (PI(3,4,5)P3-dependent Rac Exchanger) guanine nucleotide exchange factors (GEFs) are potent on-switches for Rho GTPase signalling and are frequently dysregulated in metastatic cancer2. P-Rex GEFs are autoinhibited under basal conditions and activated synergistically by binding to Gβγ and PI(3,4,5)P33. However, the molecular basis for P-Rex autoinhibition remains unknown. Here we utilise X-ray crystallography, cryo-EM and cross-linking mass spectrometry to determine the autoinhibited P-Rex1 structure. P-Rex1 forms a characteristic bipartite structure with its seven domains split into distinct N- and C-terminal modules. The two modules are connected by a previously unrecognised C-terminal four-helix bundle that binds the N-terminal Pleckstrin homology (PH) domain. In the N-terminal module, the catalytic surface of the Dbl homology (DH) domain is occluded by the compact arrangement of the PH and DEP1 domains. Structural analysis of the DH-PH-DEP1 array reveals that a remarkable conformational transition, centred on a 126° opening of a hinge helix reminiscent of calmodulin dynamics, leads to the release of DH domain autoinhibition. Furthermore, given the off-axis position of the Gβγ and PI(3,4,5)P3 binding sites, our data suggest that concurrent Gβγ and PI(3,4,5)P3 requires counter-rotation of the two halves of P-Rex1 by 90°, leading to the uncoupling of the PH domain from the four-helic¬¬al bundle, and release of the autoinhibited DH domain to drive Rho GTPase signalling.