The extensive application of plasticizers has led to significant environmental issues. This study focused on the ecotoxic effects on earthworms of the traditional plasticizer di(2-ethylhexyl) phthalate (DEHP) and non-phthalate plasticizers di(ethylhexyl) terephthalate (DEHT) and acetyltributyl citrate (ATBC). At an environmentally relevant concentration (50 mg/kg), significant accumulation of ROS was observed in earthworms, with a trend of DEHP >
DEHT >
ATBC, inducing oxidative stress and lipid peroxidation. DEHP, DEHT, and ATBC impaired the energy metabolism in earthworms, as evidenced by a sharp reduction in ATP content ranging from 43.2 % to 75.8 %, which was attributed to the disruption of glycolysis and the TCA cycle. Concurrently, the numbers of cocoons and juvenile earthworms decreased by 23.3 %-76.7 % and 24.2 %-75.8 %, respectively, indicating a significant decline in reproductive capacity. Using qPCR, AlphaFold2, and molecular docking techniques, this study is the first to report that because of their similar molecular structures, the alternatives to DEHP exhibit estrogen-like effects in earthworms, which may be a key mechanism of reproductive toxicity. These results provide valuable references and profound insights for the development of novel plasticizer alternatives and the assessment of their impact on soil ecosystems.