Premature infants are at higher risk for developing chronic lung diseases, especially following supplemental oxygen (hyperoxia) in early life. We previously demonstrated that moderate hyperoxia (<60% O(2)) induces cellular senescence in fetal airway smooth muscle cells (fASM) and fibroblasts. However, the mechanisms underlying O(2)-induced senescence are still under investigation. In this study we investigated the role of endoplasmic reticulum (ER) stress and mitochondrial dysfunction, using fASM cells exposed to 21% O(2) (normoxia) vs. ∼50% O(2) (hyperoxia). Normoxia or hyperoxia-exposed fASM were treated with the ER stress inhibitor salubrinal [12.5 μM], the antioxidant MitoQ [100 nM], or the mitochondrial fission inhibitor Mdivi-1 [10 μM]. Samples were harvested at day 2, 3, and 7 and analyzed for markers of senescence, oxidative stress, ER stress response, and mitochondrial dynamics using protein analysis and fluorescence microscopy. Hyperoxia enhanced senescence, upregulating multiple markers of DNA damage in particular, cyclin-dependent cell cycle regulator p21, cytosolic and mitochondrial reactive oxygen species (ROS) levels, mitochondria fragmentation, and anti-apoptosis B-cell lymphoma-extra large (Bcl-xL), while downregulating the proliferation marker Ki-67. Hyperoxia also activated all three ER stress pathways. However, the level of p21 and/or Bcl-xL was decreased in hyperoxia-exposed cells treated with the ER stress inhibitor salubrinal or the antioxidant MitoQ, but not the fission inhibitor Mdivi-1. These findings highlight the role of mitochondrial ROS and ER stress in hyperoxia-induced senescence of fASM and suggest that mitochondrial-targeted antioxidants and/or inhibitors of ER stress pathways can blunt the detrimental effects of hyperoxia in developing lungs.NEW & NOTEWORTHY Supplemental O(2) (hyperoxia) in premature infants detrimentally affects bronchial airways leading to increased senescence. Understanding the mechanisms by which hyperoxia initiates senescence in developing airways is critical for future therapeutic strategies. The current study showed that hyperoxia-induced senescence is mediated through increased mitochondrial reactive oxygen species and endoplasmic reticulum (ER) stress. ER stress inhibitors or mitochondria-targeted antioxidants may represent future therapies to blunt detrimental effects of supplemental oxygen in developing lungs.