The objective of this study was to evaluate the sub-lethal toxicity of hexabromocyclododecane (HBCDD) in fish. Adult Chinese rare minnows as in vivo models were exposed to waterborne HBCDD from 1 to 500 μg/l for 14, 28 and 42 days. Hepatic CYP1A1 (ethoxyresorufin- O-deethylase, EROD) and CYP2B1 (pentaoxyresorufin- O-depentylase, PROD) activities were measured. At the same time, molecular biomarkers of oxidative stress were also assayed in the brain, including reactive oxygen species (ROS), lipid peroxidation products (thiobarbituric acid-reactive substances, TBARS), DNA damage and protein carbonyl, as well as superoxide dismutase (SOD) activity and glutathione (GSH) content. DNA damage was evaluated using the Comet assay on erythrocytes. Besides, the content of HBCDD in whole fish was determined after 42 days exposure. The results show that HBCDD could induce EROD and PROD at 500 μg/l after 28 days exposure, and at 100 to 500 μg/l after 42 days exposure ( P < 0.05), respectively. ROS formation in fish brain was observed to be increased in both time- and dose-dependent manner due to HBCDD exposure. The significant increases in TBARS and protein carbonyl contents occurred in fish brain after 28 and 42 days exposure ( P < 0.05). Significant DNA damage in erythrocytes by Comet assay was also found in the 100–500 μg/l exposure groups ( P < 0.05) after 42 days exposure. Moreover, significant depletion in brain GSH content occurred in all treated groups ( P < 0.05) and apparent inhibition in SOD activity in brain was observed in the groups of 10–500 μg/l concentrations during 42 days exposure. The results demonstrate that increasing duration of HBCDD exposure induced EROD and PROD activities, caused excess ROS formation, finally resulted in oxidative damage to lipids, proteins and DNA and decreased antioxidant capacities in fish. Chemical analysis of HBCDD in whole fish showed accumulation up to 654 μg/g wet weight.