Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system which causes chronic demyelination. Hydroxychloroquine (HCQ) possess immunosuppressive and anti-inflammatory properties. The aim of this study was to investigate the effect of HCQ on miR-219 and miR-155-3p expression changes in MS-induced model. The animal model was induced by the administration of cuprizone containing food pellets (0.2%). Briefly, C57BL/6 mice were randomly divided into five groups. Group 1 received normal food and water during the study. Group 2 received cuprizone pellets for 5 weeks (demyelination phase) following one-week normal feeding during the remyelination phase. The remaining three groups received HCQ (2.5, 10 and 100 mg/kg) via drinking water during the demyelination phase. At the end of each phase, mice were deeply anesthetized, perfused with PBS through the heart, and their brains were removed. Brain sections stained with luxol fast blue and the images were analyzed. Also, the expression levels of miR-219 and miR-155-3p were evaluated by quantitative Real-Time PCR in all samples. HCQ decreased the expression of miR-155-3p and increased miR-219 expression in animals treated with 100 mg/kg of HCQ compared to the control group (p < 0.0001) and the cuprizone group (p < 0.0001). LFB method revealed a gradual increment of myelination in animals treated with 10 and 100 mg/kg of HCQ compared to the cuprizone group. Based on the obtained results of this study, HCQ can decrease microglial activity and increase oligodendrocye production by altering the expression of disease-associated miRNAs.