Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor regulating the expression of a battery of cytoprotective genes. Constitutive Nrf2 activation in many tumors enhances cell survival and resistance to anticancer drugs. Using a cell-based ARE-reporter assay we discovered that the flavonoid luteolin is a potent Nrf2 inhibitor. Luteolin inhibited ARE-driven gene expression redox-independently. In non-small-cell lung cancer A549 cells, which possess constitutively active Nrf2, luteolin elicited a dramatic reduction in Nrf2 at both the mRNA and the protein levels, leading to decreased Nrf2 binding to AREs, down-regulation of ARE-driven genes, and depletion of reduced glutathione. After transcription was blocked with actinomycin D, 1μM luteolin decreased the Nrf2 mRNA level by 34% in 30min, indicating its role in accelerating Nrf2 mRNA turnover. At physiological concentrations, luteolin significantly sensitized A549 cells to the anticancer drugs oxaliplatin, bleomycin, and doxorubicin. However, knockdown of Nrf2 using siRNA essentially abolished the induced sensitivity by the flavonoid, implying the importance of inhibiting Nrf2 for its activity. Our study demonstrates that an Nrf2 inhibitor can enhance the responsiveness of cancer cells to chemotherapeutic drugs and indicates the potential application of luteolin as a natural sensitizer in chemotherapy.