Hypoxia is common in aquatic environments, and exposure to hypoxia followed by reoxygenation is often believed to induce oxidative stress. However, there have been relatively few studies of reactive oxygen species (ROS) homeostasis and oxidative status in fish that experience natural hypoxia-reoxygenation cycles. We examined how exposure to acute hypoxia (2 kPa O2) and subsequent reoxygenation (to 20 kPa O2) affects redox status, oxidative damage, and antioxidant defenses in estuarine killifish (Fundulus heteroclitus), and whether these effects were ameliorated or potentiated by prolonged (28 day) acclimation to either constant hypoxia or intermittent cycles of nocturnal hypoxia (12 h normoxia: 12 h hypoxia). Acute hypoxia and reoxygenation led to some modest and transient changes in redox status, increases in oxidized glutathione, depletion of scavenging capacity, and oxidative damage to lipids in the skeletal muscle. The liver had greater scavenging capacity, total glutathione concentrations, and activities of antioxidant enzymes (catalase, glutathione peroxidase) than the muscle, and generally experienced less variation in glutathiones and lipid peroxidation. Unexpectedly, acclimation to constant hypoxia or intermittent hypoxia led to a more oxidizing redox status (muscle and liver) and it increased oxidized glutathione (muscle). However, hypoxia acclimated fish exhibited little to no oxidative damage (as reflected by lipid peroxidation and aconitase activity), in association with improvements in scavenging capacity and catalase activity in muscle. We conclude that hypoxia acclimation leads to adjustments in ROS homeostasis and oxidative status that do not reflect oxidative stress but may instead be part of the suite of responses that killifish use to cope with chronic hypoxia.