Co-administration phenoxodiol with doxorubicin synergistically inhibit the activity of sphingosine kinase-1 (SphK1), a potential oncogene of osteosarcoma, to suppress osteosarcoma cell growth both in vivo and in vitro. Elucidation of the mechanisms of chemo-resistance and implementation of strategies to overcome it will be pivotal to improve the survival for osteosarcoma (OS) patients. We here suggest that sphingosine kinase-1 (SphK1) might be the key factor contributing to chemo-resistance in OS. Our Western-blots and immunohistochemistry results showed that SphK1 is over-expressed in multiple clinical OS tissues. Over-expression of SphK1 in OS cell line U2OS promoted its growth and endorsed its resistance against doxorubicin, while knocking-down of SphK1 by shRNA inhibited U2OS cell growth and increased its sensitivity to doxorubicin. Co-administration phenoxodiol with doxorubicin synergistically inhibited SphK1 activity to trigger cellular ceramide accumulation, and achieved synergistic anti-OS growth effect, accompanied with a significant increased of apoptosis and cytotoxicity. Increased cellular level of ceramide by the co-administration induced the association between Akt and Protein Phosphatase 1 (PP1) to dephosphorylate Akt, and to introduce a constitutively active Akt (CA-Akt) restored Akt activation and diminished cell growth inhibition. Further, phenoxodiol and doxorubicin synergistically activated apoptosis signal-regulating kinase 1(ASK1)/c-jun-NH2-kinase (JNK) signaling, which also contributed to cell growth inhibition. Significantly, the role of SphK1 in OS cell growth and the synergistic anti-OS effect of phenoxodiol and doxorubicin were also seen in a mice OS xenograft model. In conclusion, our data suggest that SphK1 might be a critical oncogene of OS and co-administration phenoxodiol with doxorubicin synergistically inhibited the activity of SphK1 to suppress osteosarcoma cell growth both in vivo and in vitro.Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.