In human airway smooth muscle (hASM), mitochondrial volume density is greater in asthmatic patients compared to normal controls. There is also an increase in mitochondrial fragmentation in hASM of moderate asthmatics associated with an increase in Drp1 and a decrease in Mfn2 expression, mitochondrial fission and fusion proteins, respectively. Pro-inflammatory cytokines such TNFα contribute to hASM hyperreactivity and cell proliferation associated with asthma. However, the involvement of pro-inflammatory cytokines in mitochondrial remodeling is not clearly established. In non-asthmatic hASM cells, mitochondria were labeled using MitoTracker Red and imaged in 3-D using a confocal microscope. After 24-h TNFα exposure, mitochondria in hASM cells were more fragmented, evidenced by decreased form factor, aspect ratio and increased sphericity. Associated with increased mitochondrial fragmentation, Drp1 expression increased while Mfn2 expression was reduced. TNFα also increased mitochondrial biogenesis in hASM cells reflected by increased PGC1α expression and increased mitochondrial DNA copy number. Associated with mitochondrial biogenesis, TNFα exposure also increased mitochondrial volume density and porin expression, resulting in an increase in maximum O consumption rate. However, when normalized for mitochondrial volume density, O consumption rate per mitochondrion was reduced by TNFα exposure. Associated with mitochondrial fragmentation and biogenesis, TNFα also increased hASM cell proliferation, an effect mimicked by siRNA knockdown of Mfn2 expression and mitigated by Mfn2 overexpression. The results of this study support our hypothesis that in hASM cells exposed to TNFα mitochondria are more fragmented, with an increase in mitochondrial biogenesis and mitochondrial volume density resulting in reduced O consumption rate per mitochondrion.