Antioxidants potentially play an important role in the control and treatment of acute lung injury. In this study, for the first time, antioxidant effect of N-acetylcysteine (NAC) was evaluated in vivo when loaded in a polymeric nanoparticle. NAC-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NAC-PLGA NPs) were prepared by electrospray method (electrohydrodynamic atomization) and assessed in animals with direct exposure to lipopolysaccharide (LPS). Size, polydispersity index, and zeta potential of NAC-PLGA NPs were 197.5nm, 0.21, and -10.4, respectively. Fine particle fraction and emitted dose were 13.71% and 47.75%, respectively. Compared to the control group, pretreatment of LPS-challenged rats with NAC-PLGA NPs led to a significant increase in concentration of NAC in bronchoalveolar lavage fluid and pulmonary non-protein thiol levels as well as decrease in lung wet/dry weight ratio, protein concentrations, inflammatory cell counts, and levels of pulmonary myeloperoxidase and malondialdehyde. Additionally, the group showed that pulmonary architecture was preserved and infiltration of inflammatory cells and edema decreased, with lesser degree of alveolar congestion and hemorrhage. Pretreatment of animals with conventional NAC or empty PLGA NPs did not show a significant change in the above-mentioned injury indicators. In conclusion, NAC, when delivered in a polymeric nanoparticle formulation, shows improved efficacy in preventing LPS-induced lung injury by reducing the effects of reactive oxygen species and inflammation. Effectiveness of NAC-PLGA NPs can be attributed to the ability of nanoparticles to deliver NAC directly to the lung with increased retention and higher pulmonary concentrations of NAC in the lung.