Trifluralin is a preemergent herbicide that is applied to soil to control annual grasses and broadleaf weeds. It is widely used in cotton, Gossypium hirsutum L., production in China; however, the ecological safety of its continued use is a controversial issue. We studied the interaction of trifluralin and earthworms, Eisenia foetide Savigny (Annelida: Lumbricidae), to provide additional information for assessing the risk of trifluralin to ecological safety in soils. Contact toxicity assays established median lethal concentrations (LC₅₀) of 726.298 µg/L at 24 h, 418.783 µg/L at 48 h, and 82.007 µg/L at 72 h of exposure to trifluralin. Within 24 to 48 h of exposure to trifluralin, antioxidant activity (e.g., superoxide dismutase, catalase, peroxidase) increased in vivo, but by 72 h of exposure the activity was inhibited and, at high concentrations of trifluralin, death occurred. Based on the activity of glutathione S-transferase (GST) and multifunction oxidase (MFO), it appears that GSTs may be involved in the detoxification of trifluralin in vivo, and that MFOs may be the key detoxification enzymes involved. Earthworm degradation of trifluralin shortened the half-life of trifluralin in soil by as much as 1.78 d. These results provide useful information on the toxicity mechanism of trifluralin in earthworms, the role of earthworms in trifluralin degradation, as well as the ecological safety of trifluralin.