Scope: Food allergy is an increasing global health problem and perinatal administration of probiotic bacteria is currently under investigation in order to prevent the development of allergic diseases. Here, we investigated the impact of neonatal mono-colonization of mice with Lactobacillus casei BL23 on an oral sensitization to cow's milk. Methods and results: Mono-colonized (LC) mice were obtained by inoculating L. casei to germ free (GF) parents. Nine-week-old GF, LC, and conventional (CV) mice were orally sensitized to cow's milk with cholera toxin as adjuvant. Compared to GF and CV mice, LC mice developed higher casein-specific IgG responses. In contrast, no significant differences between GF and LC mice were observed for the humoral responses against whey proteins. Immunoblotting experiments performed on alpha S1-casein hydrolysates revealed the presence of small peptides immunoreactive with sera from LC mice but not from GF mice. After in vitro reactivation of splenocytes, secretion of IL-17 was higher in LC mice than in GF and CV mice. Conclusion: Neonatal mono-colonization by L. casei BL23 modulated the allergic sensitization toward food antigens. Furthermore, our data suggest that casein-specific humoral responses in LC mice were enhanced because of casein hydrolysis by L. casei into immunogenic peptides.