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Di-(2-ethylhexyl) phthalate (DEHP) is a plasticizer used in the polyvinyl chloride industry worldwide. DEHP exists in the aquatic environments for decades. However, the toxicological effects of DEHP to aquatic organisms have not been adequately researched. We investigated acute toxicity, oxidative damage, antioxidant enzyme activities, and gene expression patterns of antioxidant enzymes in juvenile and adult Daphnia magna exposed to DEHP. We found that the median lethal concentrations (LC50) of DEHP for juveniles exposed for 24 and 48 h were 0.83 and 0.56 mg L−1, respectively. The LC50 of DEHP in adults exposed for 24 and 48 h were 0.48 and 0.35 mg L−1. Daphnia magna that was exposed to DEHP had increased malondialdehyde levels for 24 h and lower total antioxidant capacity compared with the control. Activity levels of antioxidant enzymes superoxide dismutase and phase II detoxifying enzyme glutathione S-transferases were significantly higher upon initial exposure for 24 h, and enzyme activity was then diminished at high concentrations and prolonged exposure for 48 h. Gene expression levels of cat and gst were notably reduced or increased upon DEHP exposure. These findings suggest that DEHP can cause biochemical and physiological effects in juvenile and adult D. magna by inhibiting enzymes, an increase in lipid peroxidation levels and changes both transcription levels of enzymes (cat, gst). On the whole, juveniles and adults both responded similarly to DEHP. Our findings will contribute to the understanding of toxic mechanisms in phthalate esters and the evaluation of environmental risks in aquatic ecosystems.