Gene electrotransfer is a non-viral technique using electroporation for gene transfection. The method is widely used in the preclinical setting and results from the first clinical study in tumours have been published. However, the preclinical studies, which form the basis for the clinical trials, have mainly been performed in rodents and the body of evidence on electrode choice and optimal pulsing conditions is limited. We therefore tested plate and needle electrodes in vivo in porcine skin, which resembles human skin in structure. The luciferase (pCMV-Luc) gene was injected intradermally and subsequently electroporated. Simultaneously, studies with gene electrotransfer to porcine skin using plasmids coding for green fluorescent protein (GFP) and betagalactosidase were performed. Interestingly, we found needle electrodes to be more efficient than plate electrodes (p < 0.001) and electric field calculations showed that penetration of the stratum corneum led to much more homogenous field distribution at the DNA injection site. Furthermore, we have optimised the electric pulse regimens for both plate and needle electrodes using a range of high voltage and low voltage pulse combinations. In conclusion, our data support that needle electrodes should be used in human clinical studies of gene electrotransfer to skin for improved expression.