Aerosol oligonucleotide therapy has vast potential in pulmonary system diseases. Reactive oxygen species (ROS) play an important role in complex physiological processes such as cell signaling, apoptosis, etc. Therefore, to determine the mechanism of ROS involvement in polyethylenimine/oligonucleotide (PEI/ON) endocytosis in cells, we measured the fluorescence intensities of fluorescein isothiocyanate-labeled ON complexes with PEI and the changes in cytosolic Ca2+ concentration ([Ca2+]c) in A549 cells after hydrogen peroxide (H2O2) stimulation. Results showed that improved uptake of PEI/ON complexes was independent of the rise of [Ca2+]c in A549 cells, including the Ca2+ inflow and the release of Ca2+ from intracellular stores induced by 500 μM H2O2. However, the enhanced uptake efficiency was almost completely abolished by the calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor and the microtube depolymerized drug. CaMKII-dependent microtube polymerization may be responsible for the enhanced uptake of PEI/ON complexes in A549 cells under oxidative stress conditions. This study is useful for research aimed at improving aerosol oligonucleotide therapy in pulmonary system diseases.