We previously reported that tumour necrosis factor alpha (TNFα) can mimic classic ischemic preconditioning (IPC) in both cells and heart. However, the signalling pathways involved remain incompletely understood. One potential protective pathway could be TNFα-induced reactive oxygen species (ROS). We hypothesized that TNFα cytoprotection occurs through the generation of ROS which originate within the mitochondria. C 2C 12 myotubes were preconditioned with either a short period of hypoxia (IPC) or a low concentration of TNFα (0.5 ng/ml) prior to a simulated ischemic insult. ROS generation was evaluated on cells stained with dichlorofluorescin diacetate (DCFH-DA) by flow cytometry. The source of TNFα-induced ROS was examined with Mitotracker Red CM-H 2XRos. The bioenergetics of the mitochondria were evaluated by investigation of the respiratory parameters and the inner mitochondrial membrane potential. Pretreatment with TNFα improved cell viability compared with the simulated ischemic control (TNFα: 75 ± 1% versus 34 ± 1% for the control: p < 0.001). The ROS scavenger, N-2-mercaptopropionyl-glycine (MPG), reduced the viability of TNFα-stimulated cells to 15 ± 1% ( p < 0.001 versus TNFα). Similar results were obtained with IPC. TNFα stimulation increased ROS production mainly in the mitochondria, and this increase was abolished in the presence of MPG. Addition of TNFα to the cells increased State 2 respiration and modestly depolarised the membrane potential prior to the ischemic insult. In conclusion, TNFα-induced ROS generation can occur within the mitochondria, resulting in temporal mitochondrial perturbations which may initiate the cytoprotective effect of TNFα.