Alterations of Wnt signaling appear to be involved in the pathogenesis of osteosarcoma presenting mutations of Adenomatous Polyposis Coli and epigenetic down regulation of Wnt Inhibitory Factor 1. However, the precise role of Wnt effectors in the bone cancer progression remains unclear. We previously showed that Wnt/β-catenin/TCF activation are responsible for the repression of syndecan-2, a key modulator of apoptosis and chemosensitivity in osteosarcoma cells, suggesting a role of Wnt signaling in chemoresistance. In this study, we investigated the functional relationship between syndecan-2, Wntβ-catenin/TCF signaling and chemosensitivity in these cells. To this goal, we selected resistant osteosarcoma cells from sensitive human cell lines using repeated exposures to doxorubicin. In responsive but not in resistant-derived cells syndecan-2 expression was up regulated by doxorubicin treatment. Moreover, syndecan-2 overexpression restored the sensitivity to doxorubicin in resistant-derived cells. We found that syndecan-2 induction by doxorubicin is FoxO3a-dependent. FoxO3a overexpression resulted in increased syndecan-2 expression in sensitive and resistant-derived cells. Doxorubicin modulated FoxO3a binding on syndecan-2 gene promoter and induced FoxO-dependent inhibition of Wnt/TCF activity. Conversely, β-catenin/TCF activation impaired syndecan-2 induction by doxorubicin, indicating that Wnt signaling is competing with the action of the cytotoxic drug. However, β-catenin was also found to be required for FoxO3a activity. Consistently, DKK1 and sFRP-1 altered doxorubicin action in sensitive cells, whereas inhibition of TCF activity strongly decreased cell viability and increased sensitivity to doxorubicin in sensitive and resistant cells. TCF inhibition also increased the effect of doxorubicin treatment in an orthotopic bone tumor model in mice. Altogether, these data provide evidence that the repression of syndecan-2 by Wnt/β-catenin/TCF signaling contributes to the resistance of osteosarcoma cells to doxorubicin and suggest that TCF inhibition may represent a novel therapeutic strategy in osteosarcoma. © 2012 American Society for Bone and Mineral Research.