Beta-lactoglobulins, belonging to the lipocalin family, are a widely studied group of proteins, characterized by the ability to solubilize and transport hydrophobic ligands, especially fatty acids. Despite many reports, the mechanism of ligand binding and the functional role of these proteins is still unclear, and many contradicting concepts are often encountered in the literature. In the present paper the comparative analysis of the binding properties of beta-lactoglobulins has been performed using sequence-derived information, structure-based electrostatic calculations, docking simulations, and NMR experiments. Our results reveal for the first time the mechanism of beta-lactoglobulin ligand binding, which is completely determined by the opening-closing of EF loop, triggered by Glu89 protonation. The alkaline shift observed for Glu89 pKa in porcine beta-lactoglobulin (pKa 9.7) with respect to the bovine species (pKa 5.5) depends upon the interplay of electrostatic effects of few nearby key residues. Porcine protein is therefore able to bind fatty acids provided that the appropriate pH solution conditions are met (pH > 8.6), where the EF loop conformational change can take place. The unusually high pH of binding detected for porcine beta-lactoglobulin seems to be functional to lipases activity. Theoretical pKa calculations extended to representative beta-lactoglobulins allowed the identification of key residues involved in structurally and functionally important electrostatic interactions. The results presented here provide a strong indication that the described conformational change is a common feature of all beta-lactoglobulins.