An efficient and safe method to deliver DNA in vivo is a requirement for several purposes, such as the study of gene function and gene therapy applications. Among the different nonviral delivery methods currently under investigation, in vivo DNA electrotransfer has proven to be one of the most efficient and simple methods. This technique is a physical method of gene delivery consisting of a local application of electric pulses after injection of DNA. This technique can be applied to almost any tissue of a living animal, including tumors, skin, liver, kidney, artery, retina, cornea, or even brain, but the focus of this review will be on electrotransfer of plasmid DNA into skeletal muscle and its possible therapeutic uses for systemic diseases. Skeletal muscle is a good target for electrotransfer of DNA because of the following features: a large volume of easily accessible tissue, an endocrine organ capable of expressing several local and systemic factors, and muscle fibers as postmitotic cells have a long lifespan, which allows long-term gene expression. In this review, we will describe the main characteristics of DNA electrotransfer, including toxicity and safety issues related to this technique. We will focus on the important possible therapeutic applications of electrotransfer for systemic diseases demonstrated in animal models in the recent years, in the fields of monogenic diseases, tissue-specific diseases, metabolic disorders, immune-system-related diseases, and cancer. Finally, we will discuss the advantages and challenges of this technique.