At the air/liquid interface, cation binding specificity of alkaline earth cations, Mg2+ and Ca2+, with the biologically relevant ligand carboxylate (COO-) using vibrational sum frequency generation spectroscopy is reported. The empirical evidence strongly supports that the ionic binding strength is much stronger for Ca2+ to COO- than that for Mg2+. We conclude that at a near-neutral pH, the mechanism that governs Ca2+ binding to COO- is accompanied by commensurate deprotonation of the carboxyl headgroup. In addition, surface molecular structure and ion concentration influence the cation binding behavior at the air/liquid interface. In a 0.1 M Ca2+(aq) solution, Ca2+ initially favors forming ionic complexes in a 2:1 bridging configuration (2Ca(2+):1COO(-)) but 1:1 chelating bidentate complexes (1Ca(2+):1COO(-)) gradually emerge as secondary species as the system reaches equilibrium. As the Ca2+ concentration rises to 0.3 M, the primary complexed species exists in the 2:1 bridging configuration. Unlike Ca2+, Mg2+ at 0.1 and 0.3 M favors a solvent-separated ionic complex with COO-.