Prolonged hyperoxia exposure generates excessive reactive oxygen species (ROS) and potentially leads to oxidative injury in every organ. We have previously generated Drosophila melanogaster flies that tolerate extreme oxidative stress (90%-95% O₂), a lethal condition to naive flies, through a long-term laboratory selection. We found that hyperoxia-selected (S(O2)A) flies had a significantly longer lifespan in hyperoxia and paraquat-induced oxidative stress. Prolonged hyperoxia exposure induced a significant ROS accumulation and an increased expression of oxidative stress markers, including lipid peroxidation and protein carbonyl contents in control flies, but not in S(O2)A flies. Enzymatic assays revealed that antioxidant enzyme activity in S(O2)A flies was similar to that in control flies. However, in isolated mitochondria and using electron paramagnetic resonance, we observed that S(O2)A flies displayed a decreased superoxide yield during state 3 respiration as compared to control flies and that the activity of electron transport chain complex I and III was also inhibited in S(O2)A flies. Our observations lead to the hypothesis that decreased complex activity results in a decreased ROS production, which might be a major potential adaptive mechanism of hyperoxia tolerance.