A major issue for successful human gene therapy or genetic vaccination is a safe high-transgene expression level. Plasmid-based (non-viral) physical methods of gene transfer offered attracting approaches but their low efficiencies have limited their use in human pre-clinical trials. One of the limits appears to be the size of the plasmid that must be transferred across the cell membrane to the nucleus for its processing. In the present work to enhance gene transfer and expression, we evaluated a new generation of DNA vector; the minicircle, combined with the electropulsation technique. Minicircle is a doubled-stranded circular DNA with reduced size as it is devoid of bacterial sequences. We showed that electrotransferred minicircle encoding green fluorescent protein had higher in vitro transfection level compared with full-length plasmid. We demonstrated that minicircle great efficiency was not because of cellular toxicity decrease but was correlated to more efficient vector uptake by cells. Vector electrotransfection was operated in vivo and, using fluorescence imaging, minicircle electrotransfer was shown to enhance the efficiency and duration of tissue-targeted gene delivery and expression. By combining powerful expression and delivery systems, we have provided a valuable method for new approaches in gene therapy and genetic vaccination.