β-lactoglobulin (LG) is a major bovine milk protein, containing a central calyx and a second exosite beyond the calyx to bind vitamin D; however, the biological function of LG in transporting vitamin D remains elusive. Crystallographic findings from our previous study showed the exosite to be located at the pocket between the α-helix and β-strand I. In the present study, using site-directed mutagenesis, we demonstrate that residues Leu143, Pro144 and Met145 in the γ-turn loop play a crucial role in the binding. Further evidence is provided by the ability of vitamin D3 to block the binding of a specific mAb in the γ-turn loop. Using the mouse (n = 95) as an animal model, we initially demonstrated that LG is a major fraction of milk proteins responsible for uptake of vitamin D. Most interestingly, dosing mice with LG supplemented with vitamin D3 revealed that native LG containing two binding sites gave a saturated concentration of plasma 25-hydroxyvitamin D at a dose ratio of 2 : 1 (vitamin D3/LG), whereas heated LG containing one exosite (lacking a central calyx) gave a ratio of 1 : 1. We have demonstrated for the first time that LG has a functional advantage in the transport of vitamin D, indicating that supplementing milk with vitamin D effectively enhances its uptake.