We demonstrated that the mutant of cholera toxin (mCT) E112K which was LPS-free supported the induction of protective immunity in mucosal (e.g. lung lavage) and systemic (e.g. serum) compartments when given nasally with vaccine-grade diphtheria toxoid (DT) to mice. Significant DT-specific mucosal IgA antibody (Ab) and serum IgG, IgA and IgM Ab responses were induced when LPS-depleted mCT E112K or native CT (nCT) was co-administered nasally with DT. The analysis of DT-specific Ab-forming cell (AFC) responses supported the Ab titers and significant numbers of DT-specific IgA AFC were present in the lungs, nasal passages and submandibular glands. Furthermore, DT-specific IgG AFC in cervical lymph nodes (CLN) and the spleen were induced in mice administered with DT nasally with either mCT or nCT. The analysis of antigen-specific T cell responses revealed that increased DT-specific CD4+ T cell proliferative and Th2-type cytokine responses were induced in mice nasally-immunized with DT and the LPS-free form of mCT. The neutralization of diphtheria toxin by Abs showed that DT-specific IgG Ab responses in serum and lung lavages of mice immunized with DT and mCT were protective. Furthermore, it was shown that an IgA-enriched fraction of lung lavages possessed diphtheria toxin-specific neutralizing activity. These results are the first demonstration that nasally co-administered mCT E112K can induce DT-specific protective Ab responses in mucosal compartments (e.g. lung lavages and the lungs).