Afadin is an actin filament (F-actin) and Rap1 small G protein-binding protein encoded by the MLLT4/AF-6 gene. It is abundant at cadherin-based adherens junctions in epithelial cells, endothelial cells, and fibroblasts. It contains multiple domains and interacts with many proteins, including cell adhesion molecules and their associated molecules, and signaling molecules. Many lines of evidence show that afadin plays pleiotropic functions not only in the formation of cell junctions but also in cell polarization, migration, survival, proliferation, and differentiation. In addition, it is involved in oncogenesis and metastasis. Afadin is evolutionarily conserved from Caenorhabditis elegans to Homo sapiens. Canoe, the Drosophila melanogaster counterpart of afadin, is also localized at adherens junctions and regulates cell adhesion, cytoskeletal organization, planar cell polarity, cell differentiation, and migration. Moreover, canoe regulates asymmetric cell division of Drosophila neuroblasts. Thus, afadin/AF-6 and canoe are pivotal regulatory elements in many fundamental signaling cascades in cells.