For the development of efficient gene vector, intracellular processes such as cellular uptake, endosomal release and nuclear delivery must be overcome. Viruses have also evolved and have developed sophisticated mechanisms for controlling intracellular trafficking for the efficient delivery of their genomes to nuclei in host cells for symbiosis. In the light of these mechanisms, various kinds of artificial devices have been developed to overcome the intracellular barriers. However, in the majority of studies, variation of the transfection activity before and after the modification of devices was evaluated, and intracellular trafficking remained unclear. Therefore, it is understand to recognize which of the intracellular barrier should be intensively improved to enhance the transfection activity. To clarify the rate-limited process in the current non-viral vector, we compared the intracellular trafficking between adenovirus and LipofectAMINE PLUS. As a result, we found that difference of the transfection efficiency between adenovirus and LipofectAMINE PLUS was dominantly derived from the differences on transcription activity. Therefore it is essential to consider the regulation of the intranuclear events to improve the transfection activity of artificial vector.