Graphene oxide (GO) is the most common derivative of graphene and has been used in a large range of biomedical applications. Despite considerable progress in understanding its cytotoxicity, its potential inhalation toxicity is still largely unknown. As the pulmonary surfactant (PS) film is the first line of host defense, interaction with the PS film determines the fate of the inhaled nanomaterials and their potential toxicity. Using a coarse-grained molecular dynamics model, we reported, for the first time, a novel mechanism of toxicity caused by the inhaled GO nanosheets. Upon deposition, the GO nanosheets induce pores in the PS film and thus have adverse effects on the ultrastructure and biophysical properties of the PS film. Notably, the pores induced by GO nanosheets result in increasing the compressibility of the PS film, which is an important indication of surfactant inhibition. In vitro experiments have also been conducted to study the interactions between GO and animal-derived natural PS films, qualitatively confirming the simulation results.