The three-dimensional structure of holo S100B from bovine brain has been determined at 2.0 Å resolution by X-ray diffraction. The dimeric S100B molecule is formed by non-covalent interactions between large hydrophobic surface on both S100β subunits. There are two EF-hand motifs per S100β subunit, each of which binds one calcium ion. We observe, in the calcium-bound structure, dramatic changes in the conformation of the terminal helices, from the compact structure in the apo form to a more extended form upon binding calcium. This conformational change suggests a novel mode of target recognition of S100B.