Amelotin (AMTN) protein exerts a direct role on enamel biomineralization likely due to its binding affinity with calcium phosphates (Ca-Ps). However, the kinetics and molecular mechanisms of the AMTN–Ca-P interaction remain largely unknown. Here we used in situ atomic force microscopy (AFM) to directly image the surface growth of brushite (dicalcium phosphate dihydrate, DCPD, CaHPO4·2H2O) in the presence of recombinant human AMTN. Measured step movement velocities of the DCPD (010) face show that AMTN protein promotes crystal face growth only within a limited concentration range, whereas inhibition occurs outside of this range. A peptide derived from a highly conserved and potentially phosphorylated motif (SSEEL) within the AMTN protein inhibits crystal growth similar to that of the AMTN protein at low concentration. By the use of single-molecule force spectroscopy (SMFS), we directly measure the binding of the full-length AMTN and SSEEL to the DCPD (010) face. Similar rupture forces reveal that this acti...