A wide variety of tumor cells exhibit overexpression of urokinase plasminogen activator (uPA) and its receptor (uPAR). In breast cancer, expression of uPA and uPAR is essential for tumor cell invasion and metastasis. It is also known that uPA binds to uPAR and activates the RAS extracellular signal regulated kinase (ERK) signaling pathway. In our study, small interfering RNA (siRNA) was introduced to downregulate the expression of uPA and uPAR in two breast cancer cell lines (MDA MB 231 and ZR 75 1). uPA and uPAR were downregulated individually using single constructs, and in combination using a bicistronic construct driven by a CMV promoter in a pcDNA-3 mammalian expression vector. Reverse transcription PCR (RT-PCR) and Western blot analyses indicated downregulation at both the mRNA and protein levels. In vitro angiogenesis studies using conditioned medium in HMEC-1 cells indicated a decrease in the angiogenic potential of conditioned media from treated cells when compared to the controls. This decrease in angiogenic potential was remarkably higher with the bicistronic construct. Similarly, the invasive potential of these cells decreased dramatically when treated with the bicistronic construct, thereby suggesting a synergistic effect from the downregulation of both uPA and uPAR. Furthermore, when uPA and uPAR were downregulated simultaneously, the apoptotic cascade was triggered as indicated by the upregulation of both initiator and effector caspases as well as other pro-apoptotic molecules. A mitochondrial permeability assay and FACS analysis revealed an increase in apoptotic cells in the uPA/uPAR treatment as compared to the other treatments. This overexpression of pro-apoptotic caspases in relation to the RNAi-induced downregulation of uPA and uPAR clearly suggests the involvement of the uPA-uPAR system in cell survival and proliferation in addition to their role in tumor progression.