Lead (Pb) is one of the most severe and hazardous forms of heavy metal pollution, exerting its cytotoxicity primarily by inducing oxidative stress. The existing research focused on the effects of Pb on the redox balance of various organisms. However, the comprehension of the regulatory function of oxidative stress in Pb metabolism, particularly in insects, remains unknown. In this research, we observed that Pb exposure indeed elevated reactive oxygen species (ROS) levels and triggered oxidative stress in the midgut of fruit flies. Surprisingly, we observed a reduction in Pb contents in the bodies of Pb-fed fruit flies after treatment with oxidants, while an increase in Pb content was noted after treatment with antioxidants. The ROS influenced Pb accumulation in fruit flies by inducing the expression of metallothioneins (MTs), particularly MtnB. Further study shown that ROS-induced MT expression in fruit flies is mediated by a network involving the MTF-1, JNK, and Nrf2 pathways. This insight offers a new perspective on the crosstalk between oxidative stress and metal metabolism, enhances our understanding of how insects respond to metal toxicity and the environmental implications of metal pollution.