The relationship between hydrophobicity and the protective effect of whey protein hydrolysates (WPHs) against oxidative stress was studied. Whey protein was first hydrolysed by pepsin and trypsin to obtain WPHs. After absorbed by macroporous adsorption resin DA201-C, three fractions named as M20, M40, and M60 were eluted by various concentrations of ethanol. The hydrophobicity showed a trend of increase from M20 to M60. Antioxidant ability test in vitro indicated that all the three components of WPHs displayed reasonably good antioxidant ability. Moreover, with the increase of hydrophobicity, antioxidant ability of WPHs improved significantly. Then rat pheochromocytoma line 12 (PC12) cells oxidative model was built to evaluate the suppression of oxidative stress of three components on PC12 cells induced by H2O2. Morphological alterations, cell viability, apoptosis rate, and intracellular antioxidase system tests all indicated that WPHs exert significant protection on PC cells against H2O2-induced damage. Among them, M60 had the highest protective effect by increasing 19·3% cell survival and reducing 28·6% cell apoptosis. These results suggested hydrophobicity of WPHs was contributing to the antioxidant ability and the protective effect against oxidative damage.