Ewing sarcoma is the second most common bone tumor in childhood. Despite aggressive chemotherapy and radiotherapy, the prognosis of metastatic disease remains poor. In a nude mouse model of Ewing tumor xenografts, we recently showed that human type I interferons (IFNs) inhibit the growth of established xenografts. Combined therapy with human IFNs and ifosfamide (IFO), an alkylating agent widely used in high-dose chemotherapy of Ewing tumors, results in a strong synergistic antitumor effect. We have investigated the effect of IFNs/IFO treatment on the expression of vascular endothelial growth factor (VEGF), matrix metalloproteinase 9 (MMP-9), and urokinase plasminogen activator receptor (uPAR), three key mediators of tumor growth and angiogenesis, in tumor xenografts generated either from a primary tumor (EW7) or from a metastatic tumor (COH). COH tumors expressed 5-fold higher levels of VEGF than EW7 tumors. IFNs/IFO treatment reduced by >70% the amount of VEGF in COH and EW7 tumors. We did not detect constitutive MMP-9 activity in EW7 tumors. In contrast, the metastasis-derived COH tumor expressed very high levels of active MMP-9. Although the total amount of MMP-9 remained unchanged, active MMP-9 was reduced by up to 75% in IFNs/IFO-treated COH tumors. IFNs/IFO treatment triggered in both COH and EW7 tumors the downregulation of uPAR expression, a molecule involved in vascularization and endothelial cell migration. Our results partly explain the mechanism of tumor growth inhibition by IFNs/IFO therapy and provide a rational foundation for the development of a new therapeutic approach to Ewing tumors resistant to conventional chemotherapy.