We have solved the high-resolution X-ray structure of 14-3-3 bound to two different phosphoserine peptides, representing alternative substrate-binding motifs. These structures reveal an evolutionarily conserved network of peptide–protein interactions within all 14-3-3 isotypes, explain both binding motifs, and identify a novel intrachain phosphorylation-mediated loop structure in one of the peptides. A 14-3-3 mutation disrupting Raf signaling alters the ligand-binding cleft, selecting a different phosphopeptide-binding motif and different substrates than the wild-type protein. Many 14-3-3: peptide contacts involve a C-terminal amphipathic α helix containing a putative nuclear export signal, implicating this segment in both ligand and Crm1 binding. Structural homology between the 14-3-3 NES structure and those within IκBα and p53 reveals a conserved topology recognized by the Crm1 nuclear export machinery.