Amino-acid-based surfactants are promising collectors for the flotation of calcium-bearing minerals. In this study, N-hexadecanoylglycine(Cl6Gly) was used as a collector in the flotation system of fluorapatite (FA) and dolomite. Microflotation tests using single minerals implied that Cl6Gly could be applied as an efficient collector for FA over dolomite at pH 9 and 11. Zeta potential analyses indicated that the Cl6Gly anion was preferentially adsorbed onto the FA surface at pH 9. Density functional theory calculations revealed that the Cl6Gly ion could bind to two metal atoms on the dolomite surface in a bidentate fashion. On the other hand, the Cl6Gly ion interacted with two calcium atoms on the FA surface to generate tridentate binding structures. In these tridentate binding models, hydrogen bonding between the NH of Cl6Gly and PO4 groups occurred on the FA surface. Adsorption of the Cl6Gly ion on the FA surface through tridentate binding was significantly favored over bidentate binding to dolomite surface. Hence, FA could be efficiently floated by Cl6Gly rather than dolomite.