The whey protein has a highly nutritional value but poor functional properties. Ultra-high pressure (UHP) is a new technology for food processing. It dose not cause environmental pollution and could maintain food nutrition as much as possible. The effects of applied pressure and treatment time on functional properties and conformation changes of whey protein isolated (WPI) treated by UHP were studied in present paper. The results indicated that WPI treated by UHP showed reduced solubility, foaming and emulsifying property with the increasing of applied pressure and prolonging of treatment time. For WPI treated at 400MPa for 15min, the 1-anilinonaphthalene-8-sulphonate (ANS) studies showed the increasing fluorescence regions when the applied pressure increased. The most fluorescence regions were exposed to aqueous solution for WPI treated at 400MPa for 5min, and degree of exposure reduced when treatment time was prolonged. With the increasing of applied pressure and prolonging of treatment time, the amino acid residues having ultraviolet absorbance showed the same change as hydrophobic group.