Human transthyretin (TTR) is a homotetrameric protein involved in several amyloidoses. Zn2+ enhances TTR aggregation in vitro, and is a component of ex vivo TTR amyloid fibrils. We report the first crystal structure of human TTR in complex with Zn2+ at pH 4.6–7.5. All four structures reveal three tetra-coordinated Zn2+-binding sites (ZBS 1–3) per monomer, plus a fourth site (ZBS 4) involving amino acid residues from a symmetry-related tetramer that is not visible in solution by NMR. Zn2+ binding perturbs loop E-α-helix-loop F, the region involved in holo-retinol-binding protein (holo-RBP) recognition, mainly at acidic pH; TTR affinity for holo-RBP decreases ∼5-fold in the presence of Zn2+. Interestingly, this same region is disrupted in the crystal structure of the amyloidogenic intermediate of TTR formed at acidic pH in the absence of Zn2+. HNCO and HNCA experiments performed in solution at pH 7.5 revealed that upon Zn2+ binding, although the α-helix persists, there are perturbations in the resonances of the residues that flank this region, suggesting an increase in structural flexibility. While stability of the monomer of TTR decreases in the presence of Zn2+, which is consistent with the tertiary structural perturbation provoked by Zn2+ binding, tetramer stability is only marginally affected by Zn2+. These data highlight structural and functional roles of Zn2+ in TTR-related amyloidoses, as well as in holo-RBP recognition and vitamin A homeostasis.