Whey proteins present encrypted biofunctional peptides that need to be released from the native protein to exert their biological activity. Antihypertensive whey peptides are the most studied ones, which can be explained by high prevalence of this chronic degenerative disease. The present study investigated whether the molecular changes occurred during the gastrointestinal digestion of a whey protein hydrolysate could modulate its va-sorelaxant potential in rat aortic rings. Spectrophotometric data and SDS-PAGE gel showed a small degree of hydrolysis during the gastric phase and intense intestinal proteolysis. RP-HPLC revealed the formation of a large peptide profile. During the simulated digestion, 198 peptides were generated and identified and, left-shifted the concentration-response curve of the endothelium-dependent vasorelaxation, as recorded for the digested hy-drolysates. In conclusion, gastrointestinal digestion of the whey hydrolysate leads to the generation of bioactive peptides with enhanced vasodilatory potency, reinforcing the relevance of whey-derived products in blood pressure regulation.