The nonviral gene transfer technologies include naked DNA administration, electrical or particle-mediated transfer of naked DNA, and administration of DNA-synthetic macromolecule complex vectors. Each method has its advantage, such as low immunogenicity, inexpensiveness, ease in handling, etc., but the common disadvantage is that the transfection efficiency has been relatively poor as far as conventional plasmid vectors are involved. To improve the nonviral gene transfer systems, Epstein-Barr virus (EBV)-based plasmid vectors (also referred to EBV-based episomal vectors) have been employed. These vectors contain the EBNA1 gene and oriP element that enable high transfer efficiency, strong transgene expression and long term maintenance of the expression. In the current article, I review recent preclinical gene therapy studies with the EBV plasmid vectors conducted against various diseases. For gene therapy against malignancies, drastic tumor suppression was achieved by gancyclovir administrations following an intratumoral injection with an EBV plasmid vector encoding the HSV1-TK suicide gene. Equiping the plasmid with carcinoembryonic antigen (CEA) promoter sequences enabled targeted killing of CEA-positive tumor cells, which was not accomplished by conventional plasmid vectors without the EBV genetic elements. Transfection with an apoptosis-inducing gene was also effective in inhibiting tumors. Interleukin (IL)-12 and IL-18 gene transfer, either local or systemic, induced therapeutic antitumoral immune responses including augmentation of the cytotoxic T lymphocyte (CTL) and natural killer (NK) activities, while an autologous tumor vaccine engineered to secrete Th1 cytokines via the EBV system also induced growth retardation of tumors. Non-EBV conventional plasmids were much less effective in eliciting these therapeutic outcomes. Intracardiomuscular transfer of the β-adrenergic receptor gene induced a significant elevation in cardiac output in cardiomyopathic animals, suggesting the usefulness of the EBV system in treating heart failure. The EBV-based nonviral delivery also worked as genetic vaccine that triggered prophylactic cellular and humoral immunity against acute lethal viral infection. All the nonviral delivery vehicles so far tested showed an improved transfection rate when combined with the EBV-plasmids. Collectively, the EBV-based plasmid vectors may greatly contribute to nonviral gene therapy against a variety of disorders, including malignant, congenital, chronic and infectious diseases.