An efficient and safe method to deliver DNA in vivo is a requirement for several purposes, such as study of gene function and gene therapy applications. Among the different non-viral delivery methods currently under investigation, in vivo DNA electrotransfer has proven to be one of the most efficient and simple. This technique is a physical method of gene delivery consisting in local application of electric pulses after DNA injection. Although this technique can be applied to almost any tissue of a living animal, including tumors, skin, liver, kidney, artery, retina, cornea or even brain, this review will focus on electrotransfer of plasmid DNA into skeletal muscle and its possible uses in gene therapy, vaccination, or functional studies. Skeletal muscle is a good target for electrotransfer of DNA as it is: a large volume easily accessible, an endocrine organ capable of expressing several local and systemic factors, and muscle fibres as post-mitotic cells have a long lifespan that allows long-term gene expression. In this review, we describe the mechanism of DNA electrotransfer, we assess toxicity and safety considerations related to this technique, and we focus on important therapeutic applications of electrotransfer demonstrated in animal models in recent years.