Objective Prone positioning has been shown to improve oxygenation in patients with lung injury. We hypothesized that prone positioning of lungs during ex vivo lung perfusion (EVLP) can not only improve oxygenation but also diminish ischemia–reperfusion injury (IRI). The aim of our study was to evaluate the potential benefits of prone positioning of lungs during EVLP compared with the standard supine position. Methods Ten pigs were kept in the supine position at room temperature for 2 hours after circulatory death after which lungs were procured and subjected to 5 hours of cold storage. Lungs then underwent 2 hours of cellular EVLP with either supine positioning (Control group, n = 5) or prone positioning (Prone group, n = 5). Lung function was evaluated by assessment of physiological parameters and tissue histology and cytokines. Results IRI in the Prone group was significantly less than in the Control group. Lungs in the Prone group were significantly associated with greater partial pressure of oxygen/fraction of inspired oxygen ratio median (minimum-maximum) (301 mm Hg [272-414 mm Hg] vs 166 mm Hg [109-295] mm Hg, P = .03), better static compliance (38.9 mL/cmH2O [31.1-44.3 mL/cmH2O] vs 21.5 mL/cmH2O [12.2-33.3 mL/cmH2O], P = .03), lower lung weight ratio (1.26 [1.24-1.41] vs 1.48 [1.36-2.34], P = .02), and lower interleukin-1β levels (1.6 ng/mL [0.9-5.3 ng/mL] vs 7.5 ng/mL [5.0-16.1 ng/mL], P = .04) compared with lungs in the Control group. Conclusions These data suggest that prone positioning of lungs during EVLP may diminish IRI during EVLP and improve lung function.