Background：There is an increasing attention on miRNAs because of their functional effect on polarization of CD4+ T cells. This study explores the mechanism of miR-493-5p regulating Th9 cell differentiation in allergic asthma. Methods: The allergic airway inflammation is induced by Ovalbumin(OVA)in mice. CD4+ T cells from normal mice are cultured under Th9 cell conditions. IL-9 levels in mice and CD4+T cells are analyzed by RT-qPCR, ELISA, flow cytometry and western blot. The miR-493-5p levels in mice and cells are detected by RT-qPCR. The interaction between FOXO1 and miR-493-5p is predicted by TargetScan and confirmed by dual luciferase assay. The pathological state is evaluated by H&E staining and Lung resistance is measured in the allergic mice treated with miR-493-5p agomiR before stimulation. Results: The miR-493-5p expression in OVA-induced mice decreases significantly, accompanied by a significant upregulation in IL-9, IRF4 and FOXO1 expression and proportion of CD4+Th9 cells. MiR-493-5p mimic inhibits the expression of IL-9, IRF4 and FOXO1 and Th9 cell differentiation, while the inhibitor promotes these effects. MiR-493-5p mimic represses FOXO1 expression through interacting with 3’UTR of FOXO1 mRNA. The rescue experiment proves that miR-493-5p regulates the differentiation of Th9 cell and the expression of IL-9 by targeting FOXO1. In addition, we find that miR-493-5p agomiR treatment inhibits the FOXO1, IL-9 and IRF4 expression, decreases the proportion of CD4+ Th9 cells, alleviates the pathological state of lung tissue and airway hyperreactivity in OVA-induced asthma mice. Conclusions: Our study confirmed that miR-493-5p inhibited Th9 cell differentiation in allergic asthma by targeting FOXO1.