Mitochondria are dynamic organelles, capable of altering their morphology and function. However the mechanisms governing these changes are not fully elucidated, particularly in muscle cells. We demonstrated that oxidative stress with H2O2 resulted in a 41% increase in fragmentation of the mitochondrial reticulum in myoblasts within 3 hours of exposure, an effect that was preceded by a reduction in membrane potential. Using live-cell imaging, we monitored mitochondrial motility and found that oxidative stress resulted in a 30% reduction in the average velocity of mitochondria. This was accompanied by parallel reductions in both organelle fission and fusion. The attenuation in mitochondrial movement was abolished by the addition of N-acetylcysteine. To investigate whether H2O2-induced fragmentation was mediated by Drp1, we incubated cells with mDivi1, an inhibitor of Drp1 translocation to mitochondria. mDivi1 attenuated oxidative stress-induced mitochondrial fragmentation by 27%. Moreover, we demonstrated that exposure to H2O2 upregulated ER-unfolded protein response markers prior to the initiation of mitophagy signaling and the mitochondrial-unfolded protein response. These findings indicate that oxidative stress is a vital signaling mechanism in the regulation of mitochondrial morphology and motility.