The iron binding and release of serum transferrin are pH-dependent and accompanied by a conformational change between the iron-bound (holo-) and iron-free (apo-) forms. We have determined the crystal structure of apo-hen serum transferrin (hAST) at 3.5Å resolution, which is the first reported structure to date of any full molecule of an apo-serum transferrin and studied its pH-dependent iron release by UV–vis absorption and near UV-CD spectroscopy. The crystal structure of hAST shows that both the lobes adopt an open conformation and the relative orientations of the domains are different from those of apo-human serum transferrin and human apolactoferrin but similar to that of hen apo-ovotransferrin. Spectroscopic analysis reveals that in hen serum transferrin, release of the first iron starts at a pH ∼6.5 and continues over a broad pH range (6.5–5.2). The complete release of the iron, however, occurs at pH ∼4.0. The near UV-CD spectra show alterations in the microenvironment of the aromatic residues surrounding the iron-binding sites.