Abstract: The absorption and accumulation of cypermethrin by earthworms were investigated through both one-time and superimposed contamination pathways, and exposure tests were conducted to assess its toxicological effects. The results showed that cypermethrin entering the soil by both routes of contamination could be absorbed and accumulated in earthworms, and that cypermethrin entering the soil by the superimposed route of contamination was more likely to be accumulated in earthworms after 35–56 days of exposure. Cypermethrin accumulated in earthworms inhibits the activity of Na ⁺ −K ⁺ −ATP enzymes, which affects their malondialdehyde (MDA) and cytochrome P450 (CYP450) levels and induces growth inhibition in earthworms. The results of the total toxicity response values (IBR) showed that at the same concentration, the biomarker response of earthworms triggered by cypermethrin entering the soil via the superimposed contamination pathway was more significant than that via the one-time contamination pathway as the exposure time increased.