BACKGROUND: Poly(lactic-co-glycolic acid) (PLGA) has been studied for many years as a suitable drug delivery material, because of its chemical biocompatibility, total biodegradability, and non-toxic degradation products. The aim of this study was to establish the eNOS (endothelial Nitric Oxide Synthase) plasmid DNA-loaded PLGA nanosphere gene delivery system to control blood pressure in spontaneous hypertensive rat. METHODS: To conjugate eNOS plasmid DNA to PLGA, we used the Water in Oil in Water method to formulate DNA-nanosphere mixture. For physicochemical characterization, the particle size and zetapotential were measured by a light scattering spectrophotometer. Release kinetics of PLGA nanosphere was investigated in rat vascular smooth muscle cells with FITC (Fluorescein isothiocyanate) and DAPI (4',6-diamidino-2-phenylindole). Also, the blood pressure control capability of the eNOS/PLGA gene delivery system was evaluated in spontaneous hypertensive rats with 2 times intravenous injection over 10 weeks. RESULTS: The average size of PLGA nanosphere was 398+/-37 nm, and DNA loading efficiency within PLGA nanosphere was about 30~40%. With 2 times of intravenous injection, systolic blood pressure was controlled in spontaneous hypertensive rats in 10 weeks (eNOS/PLGA group 198+/-14mmHg, control group 215+/-15 mmHg, p<0.05). Nitric oxide concentrations in serum (eNOS/PLGA group 45.7+/-3.2 nM, control group 42.7+/-4.2 nM) and urine (eNOS/PLGA group 35.7+/-2.9 nM, control group 29.9+/-4.9 nM) were increased in the eNOS/PLGA delivery group. Carotid artery and aortic relaxation was observed after the eNOS/PLGA gene delivery. CONCLUSION: In this study, the optimal condition to prepare PLGA nanosphere eNOS gene therapy was set up for a non-viral gene delivering system and the effectiveness of this model was tested in spontaneous hypertensive rats.