Currently, therapeutic Fc-fusion protein is used for treating disease. ESAT6 (6-kDa early secretory antigenic target), secreted by Mycobacterium tuberculosis, is involved in immune regulation. Administered via intranasal immunization, ESAT6-Fc fusion protein significantly alleviated allergic airway inflammation and mucus hypersecretion, reduced the proportions of Th2 cells, Th17 cells and eosinophils with no observable histopathological injury to principal organs in ovalbumin (OVA)-induced allergic asthma (AA) model mice. A analysis of the transcriptome was conducted to explore its mechanisms of immune regulation, which confirmed that ESAT6 exerts an anti-AA effect by mainly suppressing some signaling pathways activation including Natural Killer Cell-Mediated Cytotoxicity, T Cell Receptor, Th1 and Th2 Cell Differentiation, Th17 Cell Differentiation, and Chemokine, which all belong to immune system in organismal systems. Meanwhile, The RT-qPCR validation results of Differently Expressed genes (DEGs) including Cd28, Icos, Cd48, Cd247, Cd40l, Itgal, Itgb2 and Ccl5 from the aforementioned pathways were consistent with the RNA-seq results. This study revealed the fundamental mechanisms of immune regulation underlying the effects of ESAT6 on OVA-induced allergic asthma. More importantly, this study provides valuable insights into the application of mucosal immunotherapy in treating AA, and ESAT6-Fc has potential as a secure mucosal immunotherapy agent for AA.