Electropermeabilization is a non-viral method that can be used to transfer plasmid DNA (pDNA) into cells and tissues. According the applications and considered tissues, this safe method can be less efficient than the viral approaches. Biophysical mechanisms of gene electrotransfer are not entirely known. Contrary to small molecules that have direct and fast access to the cytoplasm, pDNA is electrophoretically pushed towards the permeabilized membrane where it forms a complex before being transferred into the cytoplasm. In order to understand the biophysical mechanisms of gene electrotransfer and in this way to improve it, we investigated the dependence of the topology of pDNA i.e. linear versus supercoiled on both pDNA/membrane interaction and gene expression. Our results revealed that: i) even if pDNA/membrane interactions are only slightly affected by the topology of pDNA, ii) gene transfer and expression are strongly influenced by it. Indeed, the linearization of pDNA leads to a decrease in the transfection level.