Benzo(a)pyrene (B[a]P), one of the most persistent organic pollutants, is gradually accumulating in the soil and becoming a serious threat to soil environment. However, the long-term toxic effects of B[a]P on soil organisms including earthworms remains unknown. Responses of earthworms to the one-time or multiple-time superimposed B[a]P pollution are still unclear. Using multiple-time superimposed and one-time pollution methods, the effect of B[a]P on glutathione-S-transferease(GST) activity in earthworms was investigated. Results showed that the content of the available B[a]P in the soil and the B[a]P quantity in earthworms sharply declined in the first 28 d (0-28 d) and then slowly decreased between 28 and 56 d in both the multiple-time and one-time addition methods. The GST activity in earthworms sharply declined in the first stage (1-28 d) and then slowly decreased between 28 and 56 d in both addition methods. A significantly positive correlation (p<0.01) between the quantity of B[a]P accumulated and the GST activity in earthworms was detected. Moreover, the available B[a]P in soil and the B[a]P accumulated in earthworms in multiple-time addition treatment were ranged from 13.34% to 27.98% and from 22.58% to 46.81%, respectively, which was lower than those in the one-time pollution treatment. The activity of GST in earthworm’s tissue cells under multiple-time superimposed pollution was 3.01% to 33.07% lower than that in one-time pollution treatment during the whole incubation time. This results indicated that the soil available B[a]P and it’s toxicity to earthworms in the multiple-time polluted soil was lower than that in the one-time polluted soil.