Pulmonary hypertension (PH) is a life-threatening disorder with high mortality rates and limited treatment options. Gene therapy is an alternative treatment strategy, yet viral vectors have inherent disadvantages including immune activation. The Sleeping Beauty (SB) transposon is a nonviral method of gene delivery that overcomes some of these drawbacks. A SB-based transposon harboring a constitutively active endothelial nitric oxide synthase (eNOS) gene was administered to Sprague-Dawley rats via tail vein injection using the carrier polyethylenimine. Two days after transposon delivery, monocrotaline (MCT) was administered to induce PH. Hemodynamic, histological, and molecular measurements were performed four weeks later. Animals coinjected with transposase showed a significant reduction in pulmonary arterial pressure (PABP, 31.67±6.03 mmHg, P<0.01), an attenuation of right ventricle (RV) to whole heart (WH) wt ratios (0.227±0.0252, P<0.05) and a decrease in the pulmonary vessel wall thickness index (36.87%, P<0.001), compared with those animals receiving the eNOS transposon and a nonfunctional transposase (PABP 44.33±4.04 mmHg; RV/WH ratio 0.280±0.01; wall thickness index 62.14%) or control animals receiving MCT injection alone (PABP 49.67±3.22 mmHg; RV/WH ratio 0.290±0.0265; wall thickness index 71.99%). The physiological improvements correlated with therapeutic gene expression, suggesting that transposon-based genetic approaches have utility in the treatment of PH.--Liu, L., Liu, H., Visner, G., Fletcher, B. S. Sleeping Beauty-mediated eNOS gene therapy attenuates monocrotaline-induced pulmonary hypertension in rats.