Liposome is the promising nanocarrier for pulmonary drug delivery and surface charge is its basic property. However, there is a lack of knowledge about relationship between the liposomal surface charge and its interaction with biological barriers in the lung. Therefore, the purpose of this research is to elucidate the influence of liposome surface charge on its in vivo fate. Firstly, liposomes with positive, negative and neutral surface charge were constructed and characterized, their compatibility towards pulmonary cells was studied. Then their interaction with different biological barriers in lung, including mucus, trachea, bronchoalveolar lavage fluid (BALF) and alveolar macrophage, were investigated. Their retention behavior in lung and systemic exposure were further explored. It was demonstrated that neutrally and negatively charged liposomes were safer than positively charged ones. In the conducting airway, liposome with positive surface charge could better enhance trachea distribution but only within 2 h. In the respiratory region, both neutrally and negatively charged liposomes presented improved mucus permeability, good stability in BALF containing pulmonary surfactant, decreased macrophage uptake, prolonged lung retention and decreased systemic exposure to other organs, with neutrally charged liposome showing superior performance than the negatively charged ones. While the positively charged liposome was not stable in lung microenvironment with aggregation observed, leading to increased alveolar macrophage uptake, thereby lower pulmonary retention and higher risk of systemic exposure. In conclusion, liposomal surface charge is a tunable formulation factor to modulate the interaction with biological barriers in the lung and thus in vivo fate of inhaled liposomes.