Liposomes are promising vectors for pulmonary drug delivery, and have been used in marketed inhalation products. Membrane fluidity is an important property of liposomes. However, the influence of liposome membrane fluidity on its interaction with pulmonary physiological barriers is still unclear and needs elucidation. Here, a series of PEGylated DPPC (1,2-dihexadecanoyl-rac-glycero-3-phosphocholine) liposomes with different membrane fluidity were prepared, and their interaction with different pulmonary physiological barriers, including the mucus permeation capacity, macrophage uptake, trachea distribution and retention behavior, was investigated. The liposomes exhibited sizes of around 100 nm, near-neutral surface charge, and the membrane fluidity increased with increasing cholesterol ratio. In vitro studies showed that the liposomes with lower membrane fluidity presented optimal mucus permeation efficiency, while those with higher membrane fluidity displayed lower macrophage uptake. An in vivo trachea distribution study revealed that liposomes with low or medium membrane fluidity exhibited enhanced trachea permeation. No significant difference in lung retention was found among these liposomes. In conclusion, the mucus permeation and macrophage phagocytosis behavior of liposomes could be well tuned by changing their membrane fluidity.