Whey protein isolates (WPI) may provide anti-inflammatory benefits to cystic fibrosis (CF), which could be mediated via peptides, as proteolytic digests of WPI enhance intracellular glutathione (GSH) concentrations. The objectives of this study were to investigate whether high hydrostatic pressure can (i) improve the in vitro digestibility of WPI; and (ii) generate low molecular weight (< 1 kDa) peptides from WPI hydrolysates that exert GSH-enhancing and anti-inflammatory properties in wild type and mutant CF transmembrane conductance regulator (CFTR) tracheal epithelial cells. Hydrostatic pressure processing enhanced the in vitro digestibility of WPI to proteolytic enzymes resulting in altered peptide profiles as assessed by CZE and GC-MS. The exposure of mutant CFTR cells to low molecular weight (< 1 kDa) peptides isolated from WPI hydrolysates exposed to pressure processing (pressurized WPI hydrolysates, pWPH), showed increased intracellular levels of reduced GSH and total GSH relative to treatment with peptides obtained from native WPI hydrolysates (nWPH). A tendency for decreased interleukin-8 secretion was associated with the pWPH and nWPH treatments in mutant CFTR cells, which was not observed in wild type cells. Hydrostatic pressure processing of whey proteins appears to enhance their impact on cellular GSH status in cells with the mutant CFTR condition.