Adenoviral vector mediated gene therapy has received extensive attention in airway disease treatment. However, the lack of the requisite coxsackie-adenovirus receptor (CAR) on the apical surface of airway epithelium and the host immune response to adenoviruses limit their in vivo application. In our study, we developed for the first time a novel formulation composed of anionic liposomes and adenoviruses (AL-Ad5) using a calcium-induced phase change method. The obtained formulation was employed to enhance the transduction efficiency of airway gene delivery. Our results indicated that primary cultured airway epithelial cells infected by AL-Ad5 displayed higher LacZ gene expression compared to naked adenovirus. Importantly, AL-Ad5 significantly improved and prolonged LacZ gene expression in murine airway tissues when delivered in vivo by intratracheal instillation. Additionally, it was found that anionic liposomes provided immunoprotection to the adenovirus from neutralizing antibody, thus slowing down the elimination of Ad5 particles meanwhile reducing the inflammatory reaction caused by the Ad5 vector. These results suggested that the combination of anionic liposomes with adenovirus may be a useful strategy to deliver therapeutic genes into the airway epithelia and is promising in clinical application.