Arsenic exposure has been shown to induce hypoxia inducible factor 1α (HIF-1α) accumulation, however the underlying mechanism remains unknown. In the present study, we tested the hypothesis that arsenic exposure triggered the interaction between NADPH oxidase and mitochondria to promote reactive oxygen species (ROS) production, which inactivate prolyl hydroxylases (PHDs) activity, leading to the stabilization of HIF-1α protein. Exposure of human immortalized liver cell line HL-7702 cells to arsenite induced HIF-1α accumulation in a dose-dependent manner, which was abolished by SOD mimetic MnTMPyP. Inhibition of NADPH oxidase with diphenyleneiodonium chloride (DPI) or inhibition of mitochondrial respiratory chain with rotenone significantly blocked arsenite-induced ROS production, and the mitochondria appeared to be the major source of ROS production. Arsenite treatment inhibited HIF-1α hydroxylation by prolyl hydroxylases (PHDs) and increased HIF-1α stabilization, but did not affect HIF-1α mRNA expression and Akt activation. Supplementation of ascorbate or Fe(II) completely abolished arsenite-induced PHDs inhibition and HIF-1α stabilization. In conclusion, these results define a unique mechanism of HIF-1α accumulation following arsenic exposure, that is, arsenic activates NADPH oxidase-mitochondria axis to produce ROS, which deplete intracellular ascorbate and Fe(II) to inactivate PHDs, leading to HIF-1α stabilization.