SignificanceFormation of the hardest mineralized tissue in vertebrates, tooth enamel, relies on a unique set of enamel matrix proteins (EMPs). These EMPs assemble into a 3D extracellular organic matrix that directs the deposition of calcium and phosphate ions into hydroxyapatite crystallites. However, the molecular basis of EMP assembly into the organic matrix remains poorly understood. This study shows that self-assembly of the key EMPs, ameloblastin and amelogenin, involves a short linear amino acid motif that is evolutionarily conserved from the first tetrapods to man. Functionality of this motif in ameloblastin is shown to be essential for organization of the enamel organic matrix and for proper organization of hydroxyapatite crystallites into the compact bundles that determine the structure and mechanical resistance of enamel.