Abstract We have developed a novel platform for intranasal treatment of autoimmune diseases in which ADP-ribosylation determines whether immunity or tolerance is induced. Hence, cholera toxin A1-subunit based immunomodulation through the CTA1-peptide-DD fusion protein promotes enhancement, while inactive mutants induce suppression. Whereas the target population after intranasal administration of both constructs was CD103+CD11blow DC’s, both active and inactive mutants induced strong CD4 T cell priming, but they differentially affected CD4 T cell differentiation. This way inactive mutant constructs generated regulatory CD4 T cells producing IL-10 and effectively preventing experimental autoimmune encephalitis (EAE) and collagen-induced arthritis (CIA), when carrying relevant peptides. Targeted DCs expressed low levels of CD80, CD86 and CD40, while, by contrast, ADP-ribosylating CTA1-peptide-DD constructs gave significantly enhanced co-stimulation. Suppression was global in that regulatory T cells following treatment were able to suppress adoptively transferred naïve CD4 T cells and could subsequently maintain tolerance. The use of specifically tolerance-inducing fusion proteins may be a way forward in the search of effective treatments against autoimmune diseases. The immunomodulating effect on the CD103+CD11blow DCs will be described and the requirement for CTA1-binding to Gsa evaluated in detail, using Cre-lox Gsa-deficient mice.