uPAR, the three-domain membrane receptor of the serine protease urokinase, plays a crucial role in tumor growth and metastasis. Several mRNA splice variants of this receptor have been reported. One of these, uPAR-del4/5, lacking exons 4 and 5, and thus encoding a uPAR form lacking domain DII, is specifically overexpressed in breast cancer and represents a statistically independent prognostic factor for distant metastasis-free survival in breast cancer patients. The aim of the present study was to examine the molecular and cellular properties of the encoded uPAR-del4/5 protein. To investigate the impact of the uPAR-del4/5 overexpression on in vitro and in vivo aspects of tumor progression (e.g., proliferation, adhesion, invasion, metastatic seeding, and/or metastatic growth), we combined the analysis of transfected cancer cell lines with a murine xenograft tumor model. Increased expression of uPAR-del4/5 in human cancer cells led to reduced adhesion to several extracellular matrix proteins and decreased invasion through Matrigel, while cell proliferation was not affected in vitro. Moreover, invasion of uPAR-del4/5 overexpressing cells was not altered by addition of urokinase, while that of uPAR-wild-type overexpressing cells was drastically increased. Accordingly, we observed that, in contrast to uPAR-wild-type, uPAR-del4/5 does not interact with urokinase. On the other hand, when overexpressed in human breast cancer cells, uPAR-del4/5 distinctly impaired metastatic dissemination and growth in vivo. We demonstrate that the uPAR-del4/5 mRNA splice variant mediates tumor-relevant biological processes in vitro and in vivo. Our results thus illustrate how tumor-specific alternative splicing can distinctly impact the biology of the tumor.