Toxicity tests were performed to investigate possible harmful effects on medaka exposed to nano-iron. Dose-dependent decreases of superoxide dismutase (SOD) and increases of malondialdehyde (MDA) were induced in the medaka embryo, suggesting that oxidative damage was induced by nano-iron. For adult medaka, the disturbance of antioxidative balance was observed during the early exposure period based on the monitoring of the hepatic and cerebral SOD and reduced glutathione (GSH). No terminal oxidative damage occurred during the whole exposure period, probably due to the high self-recovering capability of the adult fish. Some histopathological and morphological alterations (cell swelling, hyperplasia, and granulomas, etc.) were observed in gill and intestine tissues, which confirmed that deleterious effects occurred as a result of direct contact with nano-iron. It is suggested that further evaluation should be made concerning the risk assessment of waterborne nano-iron on aquatic life.