Gene therapy has been suggested as a new treatment for acute lung injury (ALI), which is a severe inflammatory disease. Previously, amphiphilic polymeric carriers such as dexamethasone-conjugated polyethylenimine (PEI) (DP) have been used to transport plasmid DNA (pDNA) into the lungs. In the current study, hybrid nanoparticles comprising DP and cell membrane (CM) from LA-4 lung epithelial cells were developed for enhanced delivery of pDNA into the lungs. The CM components of the hybrid nanoparticles may interact with plasma membranes of target cells and facilitate intracellular uptake of pDNA. DP/CM/pDNA nanoparticles had the highest transfection efficiency into LA-4 cells at a weight ratio of 8 : 3 : 1. In vitro transfection assays showed that DP/CM/pDNA nanoparticles improved the cellular uptake and transfection efficiency of pDNA compared with PEI (25 kDa, PEI25k)/pDNA and DP/pDNA nanoparticles. The DP/CM/pDNA nanoparticles were approximately 80 nm in diameter with a zeta potential of +25 mV. To evaluate the therapeutic effects, heme oxygenase-1 pDNA (pHO-1) was administered to ALI animal models by intratracheal instillation. DP/CM/pHO-1 nanoparticles improved gene delivery efficiency compared with PEI25k/pHO-1 and DP/pHO-1 nanoparticles. As a result, inflammation in the lungs was alleviated by DP/CM/pHO-1 nanoparticles more effectively than by other nanoparticles. The results suggest that DP/CM/pDNA hybrid nanoparticles may be useful gene carriers for the treatment of ALI.