Chemical modification of whey proteins allows manipulation of their characteristics, such as surface charge and hydrophobicity. Herein, we report the influence of hydrophobization accomplished by a preacetylation stage and a subsequent combined acetylation−heating process on some characteristics of whey proteins. Hydrophobization extensively (≥90%) acetylated the available free amino groups of whey proteins. The produced protein particles were nanoscaled (75 nm) and had a significantly low isoelectric point (3.70). Hydrophobization increased the β-sheet content of whey proteins and significantly decreased the solvent exposure of tyrosine residues. It also conferred a less compact tertiary structure to the proteins and decreased the extent of disulfide-bond formation by heating. The mobility of α-lactalbumin in nonreducing electrophoresis gel increased as a consequence of hydrophobization. Then, the ability of whey proteins to catalyze hydroquinone autoxidation was examined, and it was found that the activity decreased as a result of hydrophobization. The catalytic activity of the proteins was associated with the free-amino-group content, which determined the extent of cation−π attractive interactions; ζ-potential, which determined the extent of anion−π repulsive interactions; and π-stacking between hydrophobic residues and the electron cloud of the quinone ring.