Extensive evidence implicates the urokinase plasminogen activator receptor (uPAR) in tumor growth, invasion, and metastasis. Recent studies have substantiated the importance of the interaction between uPAR and the extracellular matrix protein vitronectin (VN) for the signaling activity of the receptor in vitro, however, the possible relevance of this interaction for the activity of uPAR in tumor growth and metastasis has not been assessed. We generated a panel of HEK293 cell lines expressing mouse uPAR (muPAR(WT)), an uPAR mutant specifically deficient in VN binding (muPAR(W32A)), and a truncation variant (muPAR(ΔD1)) deficient in both VN and uPA binding. In vitro cells expressing muPAR(WT) display increased cell adhesion, spreading, migration, and proliferation associated with increased p130Cas and MAPK signaling. Disruption of VN binding or ablation of both VN and uPA binding specifically abrogates these activities of uPAR. When xenografted into SCID (severe combined immunodeficiency) mice, the expression of muPAR(WT), but not muPAR(W32A) or muPAR(ΔD1), accelerates tumor development, demonstrating that VN binding is responsible for the tumor-promoting activity of uPAR in vivo. In an orthotopic xenograft model using MDA-MB-231 cells in RAG1(-/-)/VN(-/-) mice, we document that host deficiency in VN strongly impairs tumor formation. These 2 lines of in vivo experimentation independently demonstrate an important role for VN in tumor growth even if the uPAR dependence of the effect in the MDA-MB-231 model remains to be ascertained.