Immunization of the female reproductive tract is important for protection against sexually transmitted diseases and other pathogens of the reproductive tract. However, intravaginal immunization with soluble antigens generally does not induce high levels of secretory immunoglobulin A (IgA). We recently developed safe mucosal adjuvants by genetically detoxifying Escherichia coli heat-labile enterotoxin, a molecule with a strong mucosal adjuvant activity, and here we describe the use of the nontoxic mutant LTK63 to induce a response in the mouse vagina against ovalbumin (Ova). We compared intravaginal and intranasal routes of immunization for induction of systemic and vaginal responses against LTK63 and Ova. We found that LTK63 is a potent mucosal immunogen when given by either the intravaginal or intranasal route. It induces a strong systemic antibody response and IgG and long-lasting IgA in the vagina. The appearance of vaginal IgA is delayed in the intranasally immunized mice, but the levels of vaginal anti-LTK63 IgA after repeated immunizations are higher in the intranasally immunized mice than in the intravaginally immunized mice. LTK63 also acts as a mucosal adjuvant, inducing a serum response against Ova, when given by both the intravaginal and intranasal routes. However, vaginal IgA against Ova is stimulated more efficiently when LTK63 and antigen are given intranasally. In conclusion, our results demonstrate that LTK63 can be used as a mucosal adjuvant to induce antigen-specific antibodies in vaginal secretions and show that the intranasal route of immunization is the most effective for this purpose.