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With the widespread use of diethylstilbestrol (DES), it has become a common contaminant in the aquatic environment. It is toxic to a wide range of aquatic organisms, disrupting the water flea growth and further interfering with several ecosystem services. Nevertheless, the molecular mechanism of DES in water fleas is still unexplicit. In this study, the 21-day chronic test showed that a negative effect of growth and reproduction can be observed with DES exposure. Subsequently applied transcriptomic analysis illustrated the molecular mechanism in mode freshwater invertebrate <i>Daphnia magna</i> (<i>D. magna</i>) exposed to 2, 200, and 1000 μg·L⁻¹ of DES for 9 days. Meanwhile, exposure to DES at 200 and 1000 μg·L⁻¹ significantly restrains the growth (body length) and reproduction (first spawning time) of <i>D. magna</i>. Identified differentially expressed genes (DEGs) are majorly enriched relative to energy metabolism, lipid metabolism, the digestive system, transport and catabolism pathways which were remarkably changed. These repressed and up-regulated pathways, in relation to energy synthesis and metabolism, may be the reasons for the reduced body length and delayed first spawning time. Taken together, this study revealed that DES is a threat to <i>D. magna</i> in the aquatic environment and clarifies the molecular mechanism of the toxicity.