Quercetin is the most abundant polyphenolic flavonoid found in plants. Several studies suggest that it has potent anticancer effects. The present study examines the apoptosis-inducing activity and the underlying mechanism of action of quercetin in a methotrexate (MTX)-resistant osteosarcoma model. Our results showed that quercetin inhibited cell viability in a dose-dependent manner and there was no cross-resistance between MTX and quercetin in U2-OS/MTX300 cells. The induction of apoptosis was observed by flow cyto-metry and fluorescence staining experiments. Quercetin-induced apoptosis was accompanied by a significant reduction of mitochondrial membrane potential, release of mitochondrial cytochrome c to the cytosol, activation of caspase-3, down-regulation of Bcl-2, p-Bad and up-regulation of Bax. A remarkable dephospho-rylation of Akt was also detected after quercetin treatment. Furthermore, transduction with constitutively active Akt protected against the quercetin-induced dephosphorylation of Akt and Bad as well as poly(ADP-ribose)polymerase (PARP) degradation, while combined treatment with quercetin and LY294002 enhanced the dephosphorylation of Akt, Bad and PARP cleavage in U2-OS/MTX300 cells. Taken together, our results demonstrate that quercetin-induced apoptosis in the MTX-resistant osteosarcoma cells U2-OS/MTX300 was mediated via mitochondrial dysfunction and dephosphorylation of Akt.