IL-22 is an alpha-helical cytokine which belongs to the IL-10 family of cytokines. IL-22 is produced by ROR gamma t+ innate and adaptive lymphocytes, including ILC3, gamma delta T, iNKT, Th17 and Th22 cells and some granulocytes. IL-22 receptor is expressed primarily by non-haematopoietic cells. IL-22 is critical for barrier immunity at the mucosal surfaces in the steady state and during infection. Although IL-22 knockout mice were previously shown to develop experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), how temporal IL-22 manipulation in adult mice would affect EAE course has not been studied previously. In this study, we overexpressed IL-22 via hydrodynamic gene delivery or blocked it via neutralizing antibodies in C57BL/6 mice to explore the therapeutic impact of IL-22 modulation on the EAE course. IL-22 overexpression significantly decreased EAE scores and demyelination, and reduced infiltration of IFN-gamma+IL-17A+Th17 cells into the central nervous system (CNS). The neutralization of IL-22 did not alter the EAE pathology significantly. We show that IL-22-mediated protection is independent of Reg3 gamma, an epithelial cell-derived antimicrobial peptide induced by IL-22. Thus, overexpression of Reg3 gamma significantly exacerbated EAE scores, demyelination and infiltration of IFN-gamma+IL-17A+ and IL-17A+GM-CSF+Th17 cells to CNS. We also show that Reg3 gamma may inhibit IL-2-mediated STAT5 signalling and impair expansion of Treg cells in vivo and in vitro. Finally, Reg3 gamma overexpression dramatically impacted intestinal microbiota during EAE. Our results provide novel insight into the role of IL-22 and IL-22-induced antimicrobial peptide Reg3 gamma in the pathogenesis of CNS inflammation in a murine model of MS.