Objective · To investigate the effect of miR-322-5p which targets Akt3 on Th17 differentiation in experimental autoimmune encephalomyelitis (EAE) interferedinterferon-β (IFN-β). Methods · The effect of IFN-β on EAE mice which were randomly divided into IFN-β group and PBS group was examine. The percents of Th17 in the two groups were comparedfluorescence activated cell sorting. The miRNAarray was made to find different miRNAs between those two groups. MiR-322-5p was screened for further research. The target gene of miR-322-5p was predicted using softwares and the common predicted target gene Akt3 was got. The of Akt3 was detected after IFN-β intervention and miR-322-5p over. The target relationship between Akt3 and miR-322-5p was verifiedluciferase reporter assay. At last, the effect of Akt3 on Th17 differentiation was explored in vitro. Results · Comparedto PBSgroup, thepercent of Th17 wassignificantlydownregulated, the ofmiR-322-5p wassignificantlyupregulated and Akt3 was significantly downregulated in IFN-β group. The of Akt3 was obviously decreased after overexpressing miR-322-5p. Luciferase reporter assay showed that Akt3 was directly targetedmiR-322-5p. The percent of Th17 differentiation was greatly promotedAkt3 in vitro. Conclusion · IFN-βsignificantly ameliorates the severityof EAEbyaffecting miR-322-5p whichcan inhibitTh17 differentiationtargeting Akt3.