Abstract: As a persistent organic pollutant (POP), benzo(a)pyrene (BaP) is gradually accumulating in the soil, posing a serious threat to the soil environment quality. The routine approach to environmental risk assessment of BaP is only based on one-time pollution, i.e. mixing the soil with BaP only once, which deviates from the step-by-step procedure of BaP entering the soil. In this paper, a toxic bioassay was conducted to investigate toxic effects of BaP on superoxide dismutase (SOD) and peroxidase (POD) activities and malondialdehyde (MDA) levels in coelomocytes of earthworm (Eisenia fetida), using the multiple-time superimposed and one-time pollution methods. Results show that with the exposure to BaP going on, SOD and POD activities and MDA content in coelomocytes of earthworm dropped rapidly in the first 14 days (1-14 d), and then declined slowly in the 42 days to follow (14-56 d) under both the multiple-time superimposed and one-time pollution methods. In the first 14 days, the SOD activity and MDA content in earthworms found in the 0-20 cm soil layer under superimposed pollution was 20.97% and 15.96% lower, respectively, while POD activity was 20.44% higher than that under one-time pollution. However, the SOD and POD activities, as well as the MDA content in earthworms found in the >20-40 cm soil layer under superimposed pollution was 52.89%, 18.00% and 70.60% lower than that under one-time pollution, respectively. All the above findings suggest that toxicity of multiple-time superimposed BaP pollution to earthworms was lower than that of one-time pollution. The study provides crucial data for evaluating ecological toxicity of cumulative BaP pollution in the soil.