No satisfactory treatment exists for chronic rejection (CR) after lung transplantation (LT). Our objective was to assess whether ozone (O₃) treatment could ameliorate CR. Male Sprague-Dawley inbred rats (n = 36) were randomly assigned into four groups: (1) control (n = 6), (2) sham (n = 6), (3) LT (n = 12), and (4) O₃-LT (n = 12). Animals underwent left LT. O₃ was rectally administered daily for 2 weeks before LT (from 20 to 50 μg) and 3 times/wk (50 μg/dose) up to 3 months. CR; acute rejection; and Hspb27, Prdx, Epas1, Gpx3, Vegfa, Sftpa1, Sftpb, Plvap, Klf2, Cldn5, Thbd, Dsip, Fmo2, and Sepp1 mRNA gene expression were determined. Severe CR was observed in all animals of LT group, but none of the O₃-LT animals showed signs of CR, just a mild acute rejection was observed in 1 animal. A significant decrease of Hspb27, Prdx, Epas1, Gpx3, Vegfa, Sftpa1, Sftpb, Plvap, Klf2, Cldn5, Thbd, Dsip, and Fmo2 gene expression in the O₃-LT group was observed CONCLUSIONS: O₃ therapy significantly delayed the onset of CR regulating the expression of genes involved in its pathogenesis. No known immunosuppressive therapy has been capable of achieving similar results. From a translational point of view, O₃ therapy could become a new adjuvant treatment for CR in patients undergoing LT.