Bone tumors strongly influence normal tissues and stimulate bone cells for the production of cytokines supporting proliferation and abnormal survival in cancer cells. We previously reported that the proteoglycan syndecan-2 controls the activity of various cytokines and growth factors and also modulates apoptosis and response to cytotoxic agents in osteosarcoma cell lines. Here, we show that syndecan-2 has a stronger tumor suppressor activity in vivo. We identify calpain-6 as a target gene downregulated by syndecan-2 in cells and in vivo. We demonstrate that calpain-6 expression in osteosarcoma cells depends on endothelin-1, a mediator of the tumor progression in bone. Syndecan-2 overexpression alters ERK1/2, PI3K/AKT and NF?B pathways that are calpain-6-promoting signals downstream of endothelin-1. Immunohistochemical analysis shows that calpain-6 is expressed in human bone tumors and metastases. A high expression of calpain-6 was specially found in recurrent osteosarcoma. Moreover, calpain-6 levels in primary tumors were inversely related to the response to chemotherapy. Consistently, calpain-6 was increased by doxorubicin and was found to be expressed at higher levels in doxorubicin-resistant U2OS osteosarcoma-derived cells as compared to responsive cells. Inhibition of calpain-6 with shRNA resulted in decreased proliferation, increased spontaneous apoptosis and increased sensitivity to doxorubicin and also methotrexate in responsive and resistant osteosarcoma cells. Taken together, our data show that syndecan-2 exerts its pro-apoptotic function through modulation of the endothelin-1/NF?B signaling and through downregulation of calpain-6, a protective factor that contributes to abnormal cell survival. Thus, this study identifies calpain-6 as a new possible therapeutic target in chemoresistant osteosarcoma.