The 14-3-3 proteins interact with diverse cellular molecules involved in various signal transduction pathways controlling cell proliferation, transformation, and apoptosis. To aid our investigation of the biological function of 14-3-3 proteins, we have set out to identify high-affinity antagonists. By screening phage display libraries, we have identified a set of peptides which bind 14-3-3 proteins. One of these peptides, termed R18, exhibited a high affinity for different isoforms of 14-3-3 with estimated K_D values of 7−9 × 10^-8 M. Recognition of multiple isoforms of 14-3-3 suggests the targeting of R18 to a structure that is common among 14-3-3 proteins, such as the conserved ligand-binding groove. Indeed, mutations that alter critical residues in the ligand-binding site of 14-3-3 drastically decreased the level of 14-3-3−R18 association. R18 efficiently blocked the binding of 14-3-3 to the kinase Raf-1, a physiological ligand of 14-3-3, and effectively abolished the protective role of 14-3-3 against phosphatase-induced inactivation of Raf-1. The cocrystal structure of R18 in complex with 14-3-3ζ revealed the occupancy of the general binding groove of 14-3-3ζ by R18, explaining the potent inhibitory effect of R18 on 14-3-3−ligand interactions. Such a well-defined peptide will be an effective tool for probing the role of 14-3-3 in various signaling pathways, and may lead to the development of 14-3-3 antagonists with pharmacological applications.