Acid rain has become one of the major global environmental problems, and some researches reported that acid rain may have a certain inhibition on soil biodiversity. Besides this, it is well known that earthworm (Eisenia fetida) plays an important role in the functioning of soil ecosystems. For this point, we conducted a series of experiments to investigate whether acid rain would take effects on earthworms. In the present study, the earthworms were incubated on filter paper and in soil under acid rain stress. The mortality and behavior of earthworms were recorded, and epidermal damage and the activity of the CYP3A4 enzyme were measured for the tested earthworms. Our experimental results showed that the earthworms could not survive in the acid rain stress of pH below 2.5, and acid rain with weak acidity (i.e., 4.0 ≤ pH ≤ 5.5) promoted the activity of the CYP3A4 enzyme in the earthworms, while acid rain with strong acidity (i.e., 3.0 ≤ pH ≤ 3.5) inhibited it. Moreover, the degree of damage in sensitive parts of the earthworms increased with the decrease of pH value. This study suggests that acid rain can cause discomfort response and the direct epidermal damage of earthworms, and even kill them.