Experimental autoimmune encephalomyelitis (EAE) refers to the T-helper (Th) cell-induced autoimmune disease causing demyelination, axonal loss, as well as neurodegeneration of central nervous system (CNS). EAE pathogenesis is highly dependent on T-helper 17 cells (Th17) that generate interleukin-17 (IL-17), and their activity and differentiation are tightly regulated by some cytokines and transcription factors (TFs). In the pathogenic mechanism of various autoimmune disorders, including EAE, certain miRNAs play a role. Our research detected a novel microRNA (miR) that can regulate EAE. According to the results, during EAE, the expression of miR-485 notably lowered while significant increase could be found through STAT3 expression. It was discovered that miR-485 knockdown in vivo upregulated Th17-associated cytokines while aggravating EAE, whereas miR-485 up-regulation down-regulated Th17-associated cytokines while mitigating EAE. The up-regulation of miRNA-485 in vitro inhibited Th17-associated cytokine expression within EAE CD4+ T cells. Furthermore, as revealed by target prediction and dual-luciferase reporter assays, STAT3 was miR-485s direct target, a gene that encodes a protein responsible for Th17 generation. Taken together, miRNAs exert vital functions in Th17 generation and EAE pathogenesis.