Food or water-borne enteric pathogens invade their hosts via intestinal mucosal surfaces, thus developing effective oral vaccines would greatly reduce the burden of infectious diseases. The nature of the antigen, as well as the mode of its internalization in the intestinal mucosa affects the ensuing immune response. We show that model protein antigen ovalbumin (Ova) given per-orally (p.o.) induces oral tolerance (OT), characterized by systemic IgG1-dominated antibody response, which cannot be boosted by sub-cutaneous (s.c.) immunization with Ova in complete Freund's adjuvant (CFA). Intestinal IgA generated in response to Ova feeding diminished over time and was abrogated by s.c. immunization with Ova+CFA. Humoral response to Ova was altered by administering Ova conjugated to 20 nm nanoparticles (NP-Ova). P.o. administration of NP-Ova induced systemic IgG1/IgG2c, and primed the intestinal mucosa for secretion of IgA. These responses were boosted by secondary s.c. immunization with Ova+CFA or p.o. immunization with NP-Ova. However, only in s.c.-boosted mice serum and mucosal antibody titers remained elevated for 6 months after priming. In contrast, s.c. priming with NP-Ova induced IgG1-dominated serum antibodies, but did not prime the intestinal mucosa for secretion of IgA, even after secondary p.o. immunization with NP-Ova. These results indicate that Ova conjugated to NPs reaches the internal milieu in an immunogenic form and that mucosal immunization with NP-Ova is necessary for induction of a polarized Th1/Th2 immune response, as well as intestinal IgA response. In addition, mucosal priming with NP-Ova, followed by s.c. boosting induces superior systemic and mucosal memory responses. These findings are important for the development of efficacious mucosal vaccines.