Abstract Cytokine-neuroantigen (NAg) fusion proteins have promise as a new class of antigen-specific therapies for treatment of CNS autoimmune disease. The primary objective of this study was to assess therapeutic efficacy of selected cytokine-neuroantigen fusion proteins in the Lewis rat model of EAE. The neuroantigen domain of each fusion protein was comprised of the major encephalitogenic determinant of myelin basic protein. Three cytokine-NAg fusion proteins were potent inhibitors of EAE. These fusion proteins incorporated the IL-2 cytokine as the N-terminal domain (the IL2NAg fusion protein), the secreted IL-16 cytokine as the C-terminal domain (the NAgIL16 fusion protein), or the IFN-beta cytokine as the N-terminal domain (IFNbeta-NAg fusion protein). Compared to NAg alone, these fusion proteins strongly attenuated the subsequent active induction of EAE. These fusion proteins also inhibited EAE when administered after encephalitogenic challenge during onset of EAE. In conclusion, cytokine-NAg fusion proteins that incorporate IL-2, IL-16, or IFN-beta cytokine domains may comprise an effective tolerogenic therapy for CNS autoimmune disease.