IL-22 belongs to IL-10 family cytokines and is produced by RORγt+ innate and adaptive lymphocytes, including ILC3, γδ, iNKT, Th17, and Th22 cells. IL-22 receptor, however, is expressed primarily by non-hematopoietic cells. IL-22 is critical for barrier immunity at the mucosal surfaces in the steady state and during infection. Although IL-22 knock out mice were previously shown to develop experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis (MS), how IL-22 manipulation in adult mice would affect EAE course has not been studied. In this study, we over-expressed IL-22 via hydrodynamic gene delivery or blocked it via neutralizing antibodies in C57BL/6 mice to assess the impact of IL-22 modulation on EAE course. IL-22 overexpression significantly but marginally decreased EAE scores, demyelination and reduced infiltration of IFNγ+IL-17A+ Th17 cells into central nervous system (CNS). In contrast, neutralization of IL-22 exacerbated EAE scores. Surprisingly, overexpression of Reg3γ, an epithelial cell-derived antimicrobial peptide induced by IL-22, significantly exacerbated EAE scores and infiltration of IFN-γ+IL-17A+ and IL-17A+GM-CSF+ Th17 cells to CNS. Our results provide novel insight into the role of IL-22 and IL-22-induced antimicrobial peptides in the pathogenesis of CNS inflammation in a murine model of MS.