Aristolochic acid I (AAI) is found primarily in the plant Aristolochia. Consumption of products containing AAI has been linked with permanent kidney damage and urothelial carcinoma. This study applied human proximal tubule epithelial cell line (HK-2) to examine the relationship among AAI-induced intracellular oxidative stress, DNA damage and MAP kinase activation. High concentrations of AAI caused a decrease in cell viability and an increase in the activity of caspase 3. AAI treatment also led to a dose-dependent increase of reactive oxygen species (ROS) in HK-2 cells, and the presence of antioxidant glutathione (GSH) effectively inhibited ROS generation. Stimulating HK-2 cultures with AAI also led to GSH depletion. Results from single cell gel electrophoresis (SCGE) assays demonstrated that AAI showed the ability to increase the levels of DNA strand breaks in HK-2 cells. Up-regulation of luciferase activity driven by the Nrf2 binding element was also observed after 200 μM AAI treatment. Exposure of HK-2 cells with AAI activated both ERK1/2 and p38 kinase phosphorylation, while only the MEK1/2 inhibitor, U0126, significantly decreased the levels of AAI-mediated ROS. In addition,both U0126 and SB202190 effectively reversed the levels of DNA damage triggered by AAI. This suggests that AAI treatment of HK-2 results in ROS formation and DNA damage. Furthermore, ROS generation occurs via the MEK/ERK1/2 signaling pathway, whereas DNA damage occurs via both the ERK1/2 and p38 pathways.