Outer membrane vesicles (OMVs) offer a new approach for an effective cholera vaccine. We recently demonstrated that immunization of female mice with OMVs induces a long-lasting immune response and results in protection of their neonatal offspring from Vibrio cholerae intestinal colonization. This study investigates the induced protective immunity observed after immunization with OMVs in more detail. Analysis of the stomach contents and sera of the neonates revealed significant amounts of anti-OMV immunoglobulins (Igs). Swapping of litters born to immunized and nonvaccinated control mice allowed us to distinguish between prenatal and neonatal uptakes of Igs. Transfer of Igs to neonates via milk was sufficient for complete protection of the neonates from colonization with V. cholerae, while prenatal transfer alone reduced colonization only. Detection of IgA and IgG1 in the fecal pellets of intranasally immunized adult mice indicates an induced immune response at the mucosal surface in the gastrointestinal tract, which is the site of colonization by V. cholerae. When a protocol with three intranasal immunizations 14 days apart was used, the OMVs proved to be efficacious at doses as low as 0.025 microg per immunization. This is almost equivalent to OMV concentrations found naturally in the supernatants of LB-grown cultures of V. cholerae. Heterologous expression of the periplasmic alkaline phosphatase (PhoA) of Escherichia coli resulted in the incorporation of PhoA into OMVs derived from V. cholerae. Intranasal immunization with OMVs loaded with PhoA induced a specific immune response against this heterologous antigen in mice. The detection of an immune response against this heterologously expressed protein is a promising step toward the potential use of OMVs as antigen delivery vehicles in vaccine design.