Background <p>The respiratory mucosa serves as the primary site for Pseudorabies Virus (PRV) invasion, which can elicit both local mucosal and systemic immune responses. However, due to the presence of mucosal barrier, nasal immunization with inactivated vaccines typically results in a limited immune response; thus, a mucosal adjuvant is necessary to enhance this response. Cyclic GMP-AMP (cGAMP) acts as a potent agonist of STING, exhibiting significant mucosal adjuvant properties. It plays a vital role in the regulation of innate immune responses to DNA viruses.</p> Results <p>In this study, we developed a nasal vaccine for PRV by integrating cGAMP with PRV antigen. Both mucosal and systemic antibody responses were enhanced in a dose-dependent manner by PRV antigen or cGAMP adjuvant administration. cGAMP adjuvanted PRV vaccine induced a persistent IgG but resulted in a transient IgA mucosal antibody response. Intranasal immunization with cGAMP effectively enhanced CD3⁺CD4⁺T cell responses in vivo. cGAMP adjuvanted vaccine conferred effective protection against PRV challenge. Furthermore, we also established a well differentiated air-liquid cultured mice bronchial epithelial cell (Ali-MBECs). Utilizing this model, it was demonstrated that cGAMP activates IRF3 signaling, which subsequently induced the expression of MX1, ISG56, and IFN-β.</p> Conclusion <p>This study found that cGAMP significantly enhanced the nasal immunological efficacy of the PRV inactivated vaccine by activating the STING signaling pathway within respiratory mucosa. Therefore, cGAMP demonstrated significant potential as an effective mucosal immune adjuvant to be employed in inactivated PRV vaccine. This approach offered novel technical support for the immunization and prevention against PRV infection.</p>