Chemotherapy has significantly improved the prognosis of high-grade osteosarcoma (OS), but over 30% of OS patients can still not be cured. Pemetrexed, the newly-developed anti-folate chemotherapy drug, exerted lower efficacy against OS cells. Here, we aimed to increase pemetrexed efficiency, and found that the cell-permeable short-chain ceramide (C6) significantly enhanced pemetrexed-induced viability reduction and death in cultured OS cell lines (U2OS and MG-63). Pemetrexed induced moderate apoptosis in OS cells, which was dramatically augmented by C6 ceramide. The apoptosis inhibitor z-VAD-fmk largely inhibited C6 ceramide plus pemetrexed-induced cytotoxicity and apoptosis in OS cells. By using pharmacological and siRNA-knockdown strategies, we showed that Akt-mammalian TOR (mTOR) over-activation was an important pemetrexed resistance factor in OS cells, and C6 ceramide-mediated pemetrexed sensitization effect was mediated, at least in part, by Akt-mTOR inhibition. Finally, we found that Akt-S6 Kinase 1 (S6K1, an indicator of mTOR activation) was over-activated in human OS tissues. On the other hand, the osteoblastic MC3T3-E1 cells, which expressed lower Akt-S6K1 phosphorylation, were resistant to pemetrexed and/or C6 ceramide. Together, we conclude that C6 ceramide sensitizes pemetrexed-induced apoptosis and cytotoxicity in OS cells probably through in-activation of Akt-mTOR signaling. (C) 2014 Elsevier Inc. All rights reserved.