The natural flavonoid quercetin has been suggested by epidemiological studies to have preventive activity against lung cancer; however, the mechanism of which has not been well elucidated. In this report, we demonstrate that quercetin significantly enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells. Quercetin increased expression of death receptor (DR) 5, whereas it had no effect on that of other components of the death-inducing signaling complex. Conversely, the expression of survivin was potently inhibited by quercetin. We further determined that Protein Kinase C (PKC) is essential for DR5 induction but is dispensable for suppression of survivin expression. In contrast, the blockage of the serine/threonine kinase Akt activity by quercetin is important for inhibition of survivin expression but not induction of DR5. These results suggest the pathways for regulation of DR5 and survivin expression by quercetin are distinct. Importantly, suppression of survivin-sensitized TRAIL-induced cell death and blockage of DR5 expression suppressed the synergistic cytotoxicity induced by quercetin and TRAIL co-treatment. On the whole, our data show that quercetin sensitizes TRAIL-induced cytotoxicity in lung cancer cells through two independent pathways: induction of DR5 and suppression of survivin expression, which may underlie the mechanism of the lung cancer preventive activity of quercetin. The potentiation of TRAIL-induced NSCLC cell death could be implicated in lung cancer therapy and prevention.