Food protein intake interacts with the immune system. In earlier nutritional and immunological studies, nutrients, particularly milk whey proteins, were generally administered in soluble form and by gavage. However, orogastric intubation does not represent a natural way of ingesting nutrients such as lactoferrin (Lf). We examined how different modes of oral administration of Lf could affect the regulatory effect of this molecule on intestinal and systemic immune responses. Groups of 10 female BALB/c mice were administered Lf daily for 6 wk. To address the influence of the oral modes of administration, mice were given Lf either in solution, by gastric intubation or in the drinking water, or as a powder, by buccal deposition or in the diet. Mucosal and systemic immune responses, including specific immunoglobulin (Ig) secretion, cell proliferation, and cytokine production, were analyzed and compared with those of naïve mice given water under the same conditions or positive control mice that were administered Lf by i.m. injection. The addition of Lf to the drinking water had no visible effect on the immune status. Gastric intubation, single buccal doses, and continuous doses of Lf in the diet stimulated transient systemic and intestinal antibody responses against Lf. All of these oral modes of Lf exposure biased mucosal and systemic T-cell responses toward Thelper (Th)2-types and elevated IgA production by mucosal cells. However, the less natural gastric intubation also promoted Th1-type responses as evidenced by serum IgG(2a) antibodies and the secretion of Th1 cytokine by mucosal and systemic T cells in vitro. Thus, one should carefully consider the oral mode of administration for understanding regulation of immune responses by food proteins such as Lf.