The immunomodulatory potential of fragments derived from the cow's milk allergen bovine β-lactoglobulin (BLG) was assessed in a mouse model of oral tolerance (OT) [Adel-Patient, K.; Wavrin, S.; Bernard, H.; Meziti, N.; Ah-Leung, S.; Wal, J. M. Oral tolerance and Treg cells are induced in BALB/c mice after gavage with bovine β-lactoglobulin. Allergy 2011, 66 (10), 1312-1321]. Native BLG (nBLG) and chemically denatured BLG (lacking S-S bridges, dBLG), products resulting from their hydrolysis using cyanogen bromide (CNBr) and some synthetic peptides, were produced and precisely characterized. CNBr hydrolysates correspond to pools of peptides of various sizes that are still associated by S-S bridges when derived from nBLG. nBLG, dBLG, and CNBr hydrolysate of nBLG efficiently prevented further sensitization. CNBr hydrolysate of dBLG was less efficient, suggesting that the association by S-S bridges of peptides increased their immunomodulatory potential. Conversely, synthetic peptides were inefficient even if covering 50% of the BLG sequence, demonstrating that the immunomodulatory potential requires the presence of all derived fragments of BLG and further supporting the use of partially hydrolyzed milk proteins to favor OT induction in infants with a risk of atopy.