Many fish encounter hypoxia in their native environment, but the role of mitochondrial physiology in hypoxia acclimation and hypoxia tolerance is poorly understood. We investigated the effects of hypoxia acclimation on mitochondrial respiration, O2 kinetics, emission of reactive oxygen species (ROS), and antioxidant capacity in the estuarine killifish (Fundulus heteroclitus). Killifish were acclimated to normoxia, constant hypoxia (5 kPa O2), or intermittent diel cycles of nocturnal hypoxia (12 h normoxia: 12 h hypoxia) for 28-33 days and mitochondria were isolated from liver. Neither pattern of hypoxia acclimation affected the respiratory capacities for oxidative phosphorylation or electron transport, leak respiration, coupling control, or phosphorylation efficiency. Hypoxia acclimation also had no effect on mitochondrial O2 kinetics, but P50 (the O2 tension at which hypoxia inhibits respiration by 50%) was lower in the leak state than during maximal respiration, and killifish mitochondria endured anoxia-reoxygenation without any impact on mitochondrial respiration. However, both patterns of hypoxia acclimation reduced the rate of ROS emission from mitochondria when compared at a common O2 tension. Hypoxia acclimation also increased the levels of protein carbonyls and the activities of superoxide dismutase and catalase in liver tissue (the latter only occurred in constant hypoxia). Our results suggest that hypoxia acclimation is associated with changes in mitochondrial physiology that decrease ROS production and may help improve hypoxia tolerance.