The initiation and progression of allergic asthma (AA) are associated with complex interactions between inflammation and immune response. Herein, we report the specific mechanisms underlying the molecular action of interferon (IFN)-gamma in AA regulation. We speculated that IFN-gamma inhibits Th9 differentiation by regulating the secretion of interleukin (IL)-27 from dendritic cells (DCs), thereby suppressing airway inflammation in asthma. We constructed a mouse model of ovalbumin-induced AA and overexpressed IFN-gamma to evaluate the effect on the IL-27/Th9 axis via the in vitro effect of IFN-gamma on IL-27 secretion by DCs and their influence on Th9 differentiation and asthmatic inflammation. IFN-gamma overexpression reduced the proportion of Th9 cells and DCs and altered lung morphology and cytokine production in AA-induced mice, thus suppressing the AA phenotype. In addition, exogenous IFN-gamma stimulation promoted the secretion of IL-27 and suppressed Th9 differentiation of CD4+ T cells via signal transducer and activator of transcription 1/3 (STAT1/3) signaling in a time-dependent manner. This study aimed to clarify the regulatory effect and mechanism of the IFN-gamma/DCs/IL-27/Th9 axis on AA and provide novel insights for effective targeted treatment of asthma.