14-3-3 Proteins are a ubiquitous family of molecules that participate in protein kinase signaling pathways within all eukaryotic cells. Functioning as phosphoserine/phosphothreonine-binding modules, 14-3-3 proteins participate in phosphorylation-dependent protein-protein interactions that control progression through the cell cycle, initiation and maintenance of DNA damage checkpoints, activation of MAP kinases, prevention of apoptosis, and coordination of integrin signaling and cytoskeletal dynamics. In this review, we discuss the regulation of 14-3-3 structure and ligand binding, with a focus on the role of 14-3-3 proteins in human disease, particularly cancer. We discuss the latest data on the role of different 14-3-3 isotypes, the interaction of 14-3-3 proteins with Raf, Cdc25, and various integrin family members, and the likelihood that 14-3-3 proteins could be useful therapeutic targets in the treatment of human disease.