Gene transfer using non-viral vectors is a promising approach for the safe delivery of therapeutic DNA in genetic and acquired human diseases. Whereas the lack of specific immune response favors the use of plasmid-cationic polymer complexes, the limited efficacy and short duration of transgene expression impose major hurdles in the application of non-viral gene delivery techniques. Here, we review the major cellular, metabolic and physico-chemical impediments that non-viral vectors encounter before plasmid DNA enters the nucleus. Following endocytosis of DNA-polycation complexes, a large fraction of the DNA is targeted to the lysosomes. Since the cytosolic release of heterologous DNA is a prerequisite for nuclear translocation, entrapment and degradation of plasmid DNA in endo-lysosomes constitute one of the major impediments to efficient gene transfer. Plasmid DNA that escapes the endo-lysosomal compartment encounters the diffusional and metabolic barriers of the cytoplasm, reducing greatly the number of intact plasmids that reach the nucleosol. Nuclear translocation of DNA requires either the disassembly of the nuclear envelope or active nuclear transport via the nuclear pore complex. A better understanding of the cellular and molecular basis of non-viral vector trafficking from the extracellular compartment into the nucleus may provide strategies to overcome those obstacles that limit the efficiency of gene delivery.