Mechanical ventilation (MV) with O₂-rich gas offers life-saving treatment for extremely premature infants with respiratory failure, but often leads to neonatal chronic lung disease (CLD), characterized by defective formation of alveoli and blood vessels in the developing lung. We discovered that MV of 2-4d old mice with 40%-O₂ for 8h, when compared to unventilated control pups, reduced lung mRNA expression of genes that regulate lung septation and angiogenesis (vascular endothelial growth factor-A, VEGF-A; its receptor VEGF-R2; platelet-derived growth factor-A, PDGF-A; and tenascin-C, TN-C). MV with air for 8h yielded similar results for PDGF-A and TN-C, but did not alter lung mRNA expression of VEGF or VEGF-R2. MV of 4-6d old mice with 40%-O₂ for 24h reduced lung protein abundance of VEGF-A, VEGF-R2, PDGF-A and TN-C, and resulted in lung structural abnormalities consistent with evolving CLD. After MV with 40%-O₂ for 24h, lung volume was similar to unventilated controls, whereas distal airspace size, assessed morphometrically, was greater in lungs of ventilated pups, indicative of impaired septation. Immunostaining for vimentin, which is expressed in myofibroblasts, was reduced in distal lung after 24h of MV with 40%-O₂. These molecular, cellular and structural changes occurred without detectable lung inflammation, as evaluated by histology and assays for pro-inflammatory cytokines, myeloperoxidase activity and water content in lung. Thus, lengthy MV of newborn mice with O₂-rich gas reduces lung expression of genes and proteins that are critical for normal lung growth and development. These changes yielded lung structural defects similar to those observed in evolving CLD.