Activation of the pro-apoptotic c-Jun N-terminal kinase (JNK) cell signalling pathway is initiated by binding of the small GTPase Rac1 to the intrinsically disordered scaffold protein POSH (Plenty Of SH3s).1-3 Here we identify a novel recognition mode for Rac1 binding to a non-canonical CRIB motif in the intrinsically disordered region of POSH. Using 15N nuclear relaxation rates to delineate the precise binding site, we demonstrate that the interaction involves two molecular recognition elements (MRE1 and MRE2) covering an impressive 55 amino acids of POSH. Using high-throughput crystallization screening, CrystalDirect harvesting, and automated crystal diffraction at the MASSIF-1 beamline at the ESRF, Grenoble, we obtained the crystal structure of the POSH-Rac1 complex at 1.2 Å resolution showing complete folding of both MREs of POSH upon binding to Rac1. Using an extensive set of chemical exchange saturation transfer (CEST) experiments, we map the kinetic details of the folding trajectory of POSH upon binding to Rac1. We show that the interaction initially proceeds through binding and instantaneous folding of MRE1 followed by a reversible folding event of MRE2 on the seconds timescale on the surface of Rac1. Extending our approach to the oncogenic splice variant Rac1b, which displays 100-fold lower affinity for POSH, we were able to attribute this affinity difference to a reduction in the association rate between the GTPase and POSH supporting the hypothesis that Rac1b is unable to stabilise its binding-competent closed-switch conformation.4 Taken together, our work identifies a novel recognition mode by Rac1 and has implications for understanding how Rac1b becomes defective in downstream signalling pathways and contributes to tumorigenesis in multiple human cancers.