Amelogenins are major proteins expressed by ameloblasts during development of the crown (enamel and dentin). These matrix proteins guide crystal habits of the mineral phase of developing enamel and are possible regulators of other genes/proteins during development and maturation of crown and root (dentin and cementum). This study focused on defining the effect that a specific proteolytic cleavage product of amelogenin, tyrosine-rich amelogenin peptide (TRAP), has on cementoblast behavior. Immortalized cementoblasts (OCCM-30) were exposed to TRAP in vitro. Cells treated with TRAP were evaluated for cell proliferation, gene expression for osteocalcin (OCN), osteopontin (OPN), and bone sialoprotein (BSP), and induction of mineral nodule formation. No significant difference in cell proliferation was found between vehicle-treated cells and those treated with TRAP for up to 9 d after treatment. Gene expression of OCN, OPN, and BSP in TRAP-treated cementoblasts showed down-regulation, up-regulation, and no significant change, respectively, relative to vehicle control. A marked decrease in mineral nodule formation was found in cells treated with TRAP compared with the vehicle control, in a dose-dependent manner. These data, along with our previous results demonstrating similar activity with full-length amelogenin and leucine-rich amelogenin peptide (LRAP), suggest that amelogenin-like molecules regulate mesenchymal cell behavior.