The deviant Ras activation machinery is found in approximately 30% of all human cancers. SOS1 is an important protagonist of this pathway that plays a key-role in aberrant cell proliferation and differentiation. Interaction of SOS1 with 14-3-3 proteins modulates SOS1 activity in Ras-MAPK signaling. In the present study, we analyze the 14-3-3/SOS1 protein-protein interaction (PPI) by different biochemical assays and report the high resolution crystal structure of a 13-mer motif of SOS1 bound to 14-3-3ζ. These structural and functional insights are important for the evaluation of this PPI interface for small-molecule stabilization as a new starting point for modulating the Ras-Raf-MAPK pathway.