One of the major steps limiting non-viral gene transfer efficiency is the entry of plasmid DNA from the cytoplasm into the nucleus of transfected cells. Trafficking of nuclear proteins from the cytoplasm into the nucleus through nuclear pore complexes is mediated by the presence of nuclear localization sequences (NLS) on proteins. Viral DNA and RNA also require interaction with cellular machinery for efficient nuclear import. In this article, we review the various strategies used to provide plasmid DNA with nuclear localization sequences, and discuss the possibility of developing efficient gene delivery systems based on these strategies.