Rationale: Mitochondria are dynamic organelles, which continuously change their shape through fission and fusion. Disruption of mitochondrial dynamics is involved in disease pathology through excessive reactive oxygen species (ROS) production. Accelerated cellular senescence resulting from cigarette smoke (CS) exposure with excessive ROS production has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Hence, we investigated the involvement of mitochondrial dynamics and ROS production in terms of CS extract (CSE)-induced cellular senescence in human bronchial epithelial cells (HBEC). Methods: Mitochondrial morphology was examined by electron microscopy and fluorescence microscopy. Senescence associated beta-galactosidase (SA-β-gal) staining and p21 western blotting of primary HBEC were performed to evaluate cellular senescence. Mitochondrial specific superoxide production was measured by MitoSOX staining. Mitochondrial fragmentation was induced by knockdown of mitochondrial fusion proteins (OPA1 or Mitofusins) by siRNA transfection. N-acetylcysteine (NAC), and Mito-TEMPO were used as antioxidants. Measurements and Main Results: Mitochondria in bronchial epithelial cells were prone to be more fragmented in COPD lung tissues. CSE induced mitochondrial fragmentation and mitochondrial ROS production, which were responsible for acceleration of cellular senescence in HBEC. Mitochondrial fragmentation induced by knockdown of fusion proteins also increased mitochondrial ROS production and percentages of senescent cells. HBEC senescence and mitochondria fragmentation in response to CSE treatment were inhibited in the presence of antioxidants. Conclusions: CSE-induced mitochondrial fragmentation is involved in cellular senescence through the mechanism of mitochondrial ROS production. Hence, disruption of mitochondrial dynamics may be a part of the pathogenic sequence of COPD development