Ex vivo normothermic lung perfusion (EVLP) is a novel platform and method developed to facilitate functional assessment and implementation of advanced therapies for donor lungs prior to transplantation. This study aimed to determine the safety and immunological and functional benefits of ex vivo adenoviral human interleukin-10 (AdhIL-10) gene delivery to prevent the development of primary graft dysfunction in a large animal survival model. Pig donor lungs were retrieved, preserved for 6 h at 4°C, and then randomly assigned to four groups: (1) AdhIL-10 gene therapy: 12 h EVLP + AdhIL-10 intra-bronchial delivery; (2) EVLP-control: 12 h EVLP; (3) Vector-control: 12 h EVLP + adenoviral vector intra-bronchial delivery; and (4) prolonged hypothermic preservation: additional 12 h of cold ischemia. The left lung was then transplanted and evaluated. The recipients were recovered and kept alive until day 7 post-transplant under standard triple immunosuppression. Plasma levels of hIL-10 were detected in the treatment group throughout the 7 days. Analysis of peripheral blood obtained after transplant showed no signs of hematological, renal, or hepatic toxicity in the AdhIL-10 group. The immediate post-transplant lung function was significantly better in the EVLP-control and AdhIL-10 groups. Gas exchange at day 7 was superior in allografts from the AdhIL-10 group, and the histologic inflammation score was significantly lower. Lymphocytes from AdhIL-10 group harvested from mediastinal lymph nodes at day 7 post-transplantation and co-cultured with donor lymphocytes showed significantly less interferon gamma production in an Enzyme-Linked ImmunoSpot assay when compared with non-treatment groups. It has been demonstrated in this preclinical large animal survival study that ex vivo treatment with AdhIL-10 is safe and improves post-transplant lung function over EVLP alone. Improved function and an immunological advantage in both the innate and adaptive immune responses have been demonstrated.