Ionically crosslinked nanoparticles based on high and low molecular weight chitosans (CS) were formulated with plasmid DNA or dsDNA oligomers using the ionic gelation technique with pentasodium tripolyphospate (TPP) as crosslinking agent. The resulting CS/TPP nanoparticles were investigated with regard to their physical-chemical properties, in vitro transfection efficiency, toxicity, cellular uptake, and in vivo gene expression following intratracheal administration to mice. The effects of co-formulating the nanoparticles with a model protein, BSA, were also studied. CS/TPP nanoparticles showed high encapsulation efficiencies both for plasmid DNA and dsDNA oligomers (20-mers), independent of CS molecular weight. TEM images revealed a spherical shape of the CS/TPP nanoparticles in contrast to the heterogeneous and irregular morphology displayed by conventional chitosan polyplexes. The nanoparticles showed high physical stability and no DNA release could be detected in diverse release media, nor even after incubation with heparin. Low molecular weight (LMW) CS/TPP nanoparticles gave high gene expression levels in HEK 293 cells already 2 days after transfection, reaching a plateau of sustained and high gene expression between 4 and 10 days. The inclusion of BSA into the nanostructures did not alter the inherent transfection efficiency of the nanoparticles. Confocal studies suggest endocytotic cellular uptake of the nanoparticles and a subsequent release into the cytoplasm within 14 h. LMW CS/TPP nanoparticles mediated a strong beta-galactosidase expression in vivo after intratracheal administration. The results of this study forward ionically crosslinked CS/TPP nanoparticles as a biocompatible non-viral gene delivery system and generate a solid ground for further optimization studies, for example with regard to steric stabilization and targeting.