DNA vaccines have been widely used in efforts to develop vaccines against various pathogens as well as for cancer, autoimmune diseases and allergy. DNA vaccines offer broad efficacy (particularly for their ability to generate both cellular and humoral immunity), ease of construction and manufacture and the potential for world-wide usage even in low-resource settings. However, despite their successful application in many preclinical disease models, their potency in human clinical trials has been insufficient to provide protective immunity. Nevertheless, two DNA vaccines were recently licensed for use in animals (horse and fish), underscoring the potential of this technology. Here, we describe recent advances in increasing the potency of these vaccines, in understanding their immunological mechanisms, and in their applications and efficacy in clinical trials so far.