The balance between mitochondrial fission and fusion is crucial for mitochondria to perform its normal cellular functions. We hypothesized that cigarette smoke (CS), disrupts this balance and enhances mitochondrial dysfunction in the airway. In non-asthmatic human airway smooth muscle (ASM) cells, CS extract (CSE) induced mitochondrial fragmentation and damages their networked morphology in a concentration-dependent fashion, via increased expression of mitochondrial fission protein Drp1 and decreased fusion protein Mfn2. CSE effects on Drp1 vs. Mfn2 and mitochondrial network morphology involved ROS, activation of ERK, PI3/Akt, PKC and proteasome pathways, as well as transcriptional regulation via factors such as NFB and Nrf2. Inhibiting Drp1 prevented CSE effects on mitochondrial networks and ROS generation, while blocking Mfn2 had the opposite, detrimental effect. In ASM from asthmatics, mitochondria exhibited substantial morphological defects at baseline, and showed increased Drp1 but decreased Mfn2 expression, with exacerbating effects of CSE. Overall, these results highlight the importance of mitochondrial networks and their regulation in the context of cellular changes induced by insults such as inflammation (as in asthma) or CS. Altered mitochondrial fission/fusion proteins have a further potential to influence parameters such as ROS and cell proliferation and apoptosis relevant to airway diseases.