β-lactoglobulin (β-LG), one of the most investigated proteins, is a major bovine milk protein with a predominantly β structure. The structural function of the only α-helix with three turns at the C-terminus is unknown. Vitamin D3 binds to the central calyx formed by the β-strands. Whether there are two vitamin D binding-sites in each β-LG molecule has been a subject of controversy. Here, we report a second vitamin D3 binding site identified by synchrotron X-ray diffraction (at 2.4 Å resolution). In the central calyx binding mode, the aliphatic tail of vitamin D3 clearly inserts into the binding cavity, where the 3-OH group of vitamin D3 binds externally. The electron density map suggests that the 3-OH group interacts with the carbonyl of Lys-60 forming a hydrogen bond (2.97 Å). The second binding site, however, is near the surface at the C-terminus (residues 136–149) containing part of an α-helix and a β-strand I with 17.91 Å in length, while the span of vitamin D3 is about 12.51 Å. A remarkable feature of the second exosite is that it combines an amphipathic α-helix providing nonpolar residues (Phe-136, Ala-139, and Leu-140) and a β-strand providing a nonpolar (Ile-147) and a buried polar residue (Arg-148). They are linked by a hydrophobic loop (Ala-142, Leu-143, Pro-144, and Met-145). Thus, the binding pocket furnishes strong hydrophobic force to stabilize vitamin D3 binding. This finding provides a new insight into the interaction between vitamin D3 and β-LG, in which the exosite may provide another route for the transport of vitamin D3 in vitamin D3 fortified dairy products. Atomic coordinates for the crystal structure of β-LG-vitamin D3 complex described in this work have been deposited in the PDB (access code 2GJ5). Proteins 2008. © 2007 Wiley-Liss, Inc.