Calcium binding to o-phospho-L-serine (OPS), important for calcium distribution during milk processing and for calcium bioavailability, was studied electrochemically for different conditions of temperature, pH and ionic strength in combination with quantum mechanical calculations (DFT) using L-serine (Ser) as a reference compound. Calcium binding increases strongly with increasing pH for both Ser and OPS. pH dependent binding of calcium ions was resolved using pK_a values into, respectively, K_ass,1, K_ass,2 and K_ass,3 = 20, 37 and 180 L mol⁻¹ with ΔH⁰_ass,1 ΔH⁰_ass,2 ΔH⁰_ass,3 = 16, −7.4 and −11 kJ mol⁻¹ for binding to OPS⁻, OPS²⁻ and OPS³⁻ at an ionic strength 0.2 and 15 °C. Dissolution of CaOPS at milk pH was endothermic. The shift from endothermic binding of calcium to OPS at low pH to exothermic binding at higher pH, corresponding to a shift from hydrophobic interaction (phosphate ester) to ionic binding (carboxylate/amino chelation), was supported by DFT.